Constructs and methods for enhancing protein levels in photosynthetic organisms

ABSTRACT

This invention provides novel gene constructs which enhance the efficiency of plant cells and cells of other photosynthetic organisms. Also provided are transgenic plants and seeds which overexpress proteins. Methods to elevate the amount of plastid proteins in plants and photosynthetic organisms are exemplified.

RELATED APPLICATIONS

This application is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 08/568,168, filed on Dec. 6, 1995, which is herebyincorporated in its entirety.

BACKGROUND OF THE INVENTION

All photosynthetic organisms depend on the light-harvesting reactions ofphotosynthesis for energy to produce important compounds for growth andmetabolism. Energy-rich carbohydrates, fatty acids, sugars, essentialamino acids, and other compounds synthesized by photosynthetic organismsare the basis of the food chain on which all animal life depends forexistence. Photosynthetic organisms are also the major source of oxygenevolution in the atmosphere, recycling carbon dioxide in the process.Thus life on earth is reliant on the productivity of photosyntheticorganisms, especially plants.

Plant productivity is limited by the amount of resources available andthe ability of plants to harness these resources. The conversion oflight to chemical energy requires a complex system which combines thelight harvesting apparatus of pigments and proteins. The value of lightenergy to the plant can only be realized when it is efficientlyconverted into chemical energy by photosynthesis and fed into variousbiochemical processes.

The thylakoid protein apparatus responsible for the photosyntheticconversion of light to chemical energy is one of the most complexmechanisms in the chloroplast and remains one of the most difficultbiological systems to study. Oxygen-evolving photosynthetic organisms,such as cyanobacteria, algae and plants, possess two photosystems, PSIand PSII, which cooperate in series to acquire electrons from H₂ O anddeliver them energetically up a gradient to NADP⁺. The photosyntheticproduction of NADPH and ATP then, in turn, feeds into all biochemicalpathways. The force driving the uphill flow of these electrons comesfrom the light energy absorbed by the 100-300 chlorophyll moleculesassociated with the two photosystems. An important pair of chlorophyll amolecules in the center of each photosystem modulates the movement ofelectrons. The remaining chlorophyll molecules are associated withproteins which in turn are organized into light gathering antennae thatsurround the reaction centers and transfer the light energy to them(Green et al. (1991) TIBS 16:181).

The capacity to absorb light, especially in shade, depends largely onthe size and organization of the light harvesting complexes (Lhc) in thethylakoid membranes. The LhcII light harvesting complex is the majorensemble of chlorophyll a/b binding protein (Cab) acting as an antennato photosystem II (PSII) and plays a key role in harvesting light forphotosynthesis (Kuhlbrandt, W. (1984) Nature 307:478). Plants arecapable of adjusting the size of the antennae in accordance with thelight intensity available for growth. In shade, the allocation ofnitrogen is shifted from polypeptides in the stroma, by decreasingribulose 1,5-bisphosphate carboxylase (Rbc or Rubisco) levels, to thethylakoidal proteins. Nitrogen redistribution is a compensating responseto low irradiance, balancing light harvesting and CO₂ fixation (Evans,J. R. (1989) Oecologia 78:9); Stitt, M. (1991) Plant, Cell andEnvironment 14:741).

In addition to the shift in the investment of nitrogen into differentproteins, photosynthetic organisms can adapt to low light conditions bymolecular reorganization of the light harvesting complexes (Chow et al.(1990) Proc. Natl. Acad. Sci. USA 87:7502; Horton et al. (1994) PlantPhysiol. 106:415; Melis, (1991) Biochim. Biophys. Acta. 1058:87). Aplant's reorganizational ability to compensate for changes in thecharacteristics of the light limits its productivity. Although amechanism is in place to adapt to low light conditions, photosynthesisin plants grown in suboptimal illumination remains significantly lowerdue to a limited capacity to generate ATP and NADPH via electrontransport (Dietz, K. J. and Heber, U. (1984) Biochim. Biophys. Acta.767:432; ibid (1986) 848:392). Under such conditions the capacity togenerate ATP and NADPH, the assimilatory force, will dictate thecapacity to reduce CO₂. When light is limiting, plants reorganize tomaximize their photosynthetic capacity; however, the ability to adapt islimited by molecular parameters ranging from gene expression to complexassembly to substrate and cofactor availability.

If productivity of a plant or other photosynthetic organism is to beincreased, methods to enhance the light-gathering capacity withoutrestricting CO₂ fixation must be developed.

SUMMARY OF THE INVENTION

The present invention provides a chimeric gene construct comprising apromoter region, a 5' untranslated region containing a translationalenhancer, DNA encoding a plastid-specific transit peptide which enhancesprotein import, a gene encoding a plastid protein, and a 3' untranslatedregion containing a functional polyadenylation signal. This constructproduces a high level of expression and importation of the functionalprotein to the site of its function.

In one embodiment of the present invention the promoter is a 35Scauliflower mosaic virus (CaNV) promoter. In another embodiment, thetranslational enhancer is from the 5' untranslated region of the peasmall subunit of ribulose-1,5-bisphosphate carboxylase. In anotherembodiment, the transit peptide is from the pea small subunit ofribulose-1,5-bisphosphate carboxylase. In a further embodiment, the geneencoding a protein is the pea cab gene, encoding a chlorophyll a/bbinding protein. In yet another embodiment, the 3' untranslated regioncontaining a functional polyadenylation signal is from the pea cab gene.

This invention also provides a method for enhancing the light harvestingcapability of a photosynthetic plant or organism comprising: preparing agene construct comprising a promoter, a 5' untranslated regioncontaining a translational enhancer, DNA encoding a plastid-specifictransit peptide which enhances protein import, DNA encoding a protein,preferably a structural gene encoding a chlorophyll a/b binding protein,and a 3' untranslated region containing a functional polyadenylationsignal; inserting the gene construct into a suitable cloning vector; andtransforming a photosynthetic plant or other photosynthetic organismwith the recombinant vector. Alternatively, the gene construct is coateddirectly on biolistic particles with which the cells are bombarded.

This invention provides a DNA construct which can increase the amount ofone or more proteins in a plastid, especially a chloroplast, or in thecells of photosynthetic prokaryotes. These constructs can alter thephotosynthetic apparatus to increase the ability of the plant to harvestlight, especially under conditions of low illumination.

This invention also provides methods of increasing the light-harvestingefficiency of photosynthesis and the yield of photosynthetic products(such as carbohydrates) in plants and other photosynthetic organisms.These methods can be used to increase the commercial value of plants andseeds, and be used to increase the yields of products produced fromfermentation and plant tissue culture operations.

This invention also provides a transgenic (TR) plant or photosyntheticorganism containing the construct described above. These transgenicplants and photosynthetic organisms have enhanced photosyntheticcapacity and enhanced growth capabilities useful for increased yield,tissue culture, fermentation and regeneration purposes. Compared towild-type (WT) plants, transgenic plants of this invention demonstrateincreased yield, enhanced pigmentation, increased carbohydrate content,increased biomass, more uniform growth, larger seeds or fruits,increased stem girth, enhanced photosynthesis, faster germination, andincreased ability to withstand transplant shock. Seeds produced fromthese plants are also provided by this invention, as well as plant partsuseful for production of regenerated plants and other derived products.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B is the nucleotide sequence of AB80 (pea type I LhcIIb gene)(SEQ ID NO:1) and the amino acid sequence (SEQ ID NO:2) encoded by SEQID NO:1.

FIG. 2 shows the construction of vector pSSTP.

FIG. 3 shows the construction of vector pRBCS-CAB.

FIG. 4 shows the construction of vector pCAMV-RBCS-CAB.

FIG. 5 shows the construction of Agrobacterium binary vector(pEND4K-CAMV-RBCS-CAB).

FIG. 6 is a restriction map of Agrobacterium binary vector pEND4K.

FIGS. 7A-7C show the steady state transgene transcript levels intransformed and wild-type tobacco plants. FIG. 7A shows transgene mRNAlevels in plants derived from T1 seeds of primary transformants. FIG. 7Bshows transgene mRNA levels from selected T1 plants with high levels oftransgene transcript that were self-crossed and subjected to segregationanalysis. The resulting homozygous lines were examined in the samemanner as in FIG. 7A. FIG. 7C shows the steady state Cab protein levelsin transformed (TR) and wild-type (WT) tobacco plants.

FIGS. 8A-8C show the growth and morphological characteristics (FIG. 8A)of WT and TR tobacco plants (left and right, respectively). Therespective 7th fully developed leaves from WT and TR are compared asdiagrams of whole leaves (FIG. 8B) and transverse sections (FIG. 8C).

FIG. 9 is a comparison of transgenic seedlings (top row) and controlseedlings (bottom row) after four days germination on solid MS media.

FIGS. 10A-B show a comparison of transgenic (TRA) and control (WT)tobacco callus grown for the same period of time.

FIGS. 11A-11D show electron micrographs of wild-type (WT) and transgenic(TR) mesophyll tissues (FIGS. 11A and 11C) and chloroplasts (FIGS. 11Band 11D) of tobacco leaves.

FIGS. 12A-D show a comparison of WT(∇) and TR() light response curvesfor photosynthetic oxygen evolution of plants cultivated in twodifferent light intensities: (A) 50 μmol ·m⁻² ·s⁻¹ (referred to as low);and (B) 500 μmol·m⁻² ·s⁻¹ (referred to as high).

FIGS. 13A-13D show the light response curves for qP (FIG. 13A), qN (FIG.13B), Fv/Fm (FIG. 13C), and .O slashed.O_(PSII) (FIG. 13D) measured inair for WT(∇) and TR() plants.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a DNA construct which, when incorporated intoa plant or cell of a photosynthetic organism, increases the efficiencyof plastids or a photosynthetic cell, and to methods for increasing orimproving the products of plastid metabolism via enhancement of proteinexpression and import. The present invention also relates to transgenicplants, seeds, plant cells and tissues, and other photosyntheticorganisms incorporating these constructs.

A DNA construct of this invention comprises a promoter, a 5'untranslated region containing a translational enhancer, DNA encoding aplastid-specific transit peptide which can enhance and direct import ofa gene product to a plastid or photosynthetic apparatus, a gene encodinga plastid protein, and a 3' untranslated region containing a functionalpolyadenylation signal. Insertion of this construct results in increasedexpression and importation of proteins in plastids and thephotosynthetic apparatus. These elements are usually provided asoperably-joined components in the 5' to 3' direction of transcription. Apreferred embodiment of the invention is a construct comprising a 5'constitutive promoter (such as the 35S cauliflower mosaic viruspromoter), the 5' untranslated region of pea small subunit ofribulose-1,5-bisphosphate carboxylase containing a translationalenhancer which has a nucleotide sequence consisting of residues 1 to 29of SEQ ID NO:3, DNA encoding a transit peptide which is from pea smallsubunit of ribulose-1,5-bisphosphate carboxylase, a structural geneencoding a chlorophyll a/b binding protein and a 3' untranslated regioncontaining a functional polyadenylation signal is from a pea cab gene.

This novel gene construction scheme permits simultaneous high leveltranscription, high level translation, greater mRNA stability and a highlevel of protein importation into the plastid or photosyntheticapparatus of an organism, producing overproduction of the selectedprotein (in this case, a light harvesting Cab protein) in plants andother photosynthetic organisms. The multi-level gene construction,especially the enhancement of protein importation, can be widely used toenhance the import and expression of any protein. The gene constructexemplified achieves a high level of expression and importation of thefunctional protein (chlorophyll a/b binding protein) at the site of itsfunction.

The activity of many different proteins and polypeptides involved in theprocess of photosynthesis can be enhanced by the methods of thisinvention. In addition to increased levels of endogenous proteins, theDNA constructs of this invention can be used to import and expressforeign proteins in the photosynthetic apparatus of plants and otherphotosynthetic organisms. Further, the DNA construct can contain asingle protein encoding region, or can contain additional encodingregions so that several proteins can be imported and expressed. Thus,plastids of plants and cells of photosynthetic organisms can be alteredto enhance the light-harvesting reactions of photosynthesis and/or tovary the level and kind of products of the photosynthetic darkreactions.

To produce the chimeric constructs provided in this invention, aneffective chimeric Rbcs-Cab coding region was created by combiningcoding sequences for appropriate portions of Rbcs and type I LhcIIb Cab.Transgenic tobacco plants containing the gene construct of thisinvention overproduce type I Cab of LhcIIb and possess enhanced lowlight photosynthetic activity and growth capabilities. The transgenicplants also demonstrate one or more morphological, developmental,biochemical and physiological modifications. These modifications havecommercial value in crop plants where more rapid germination and growth,higher yields and improved appearance are highly preferred. The desiredmodifications are achieved through the elevated gene expression andprotein import via this novel gene construction. Enhanced expression atthe level of de novo transcription was facilitated by attaching theRbcs-Cab gene construct to the strong CaMV 35S promoter. Furtherenhancements were obtained by increasing mRNA stability, thus increasingthe magnitude of the steady state pool of transgene transcripts. Thiswas accomplished by inclusion of the functional 3' untranslated nucleicacid sequence of the cab gene and the nucleic acid sequence encoding theRbcs transit peptide. Both nucleic acid sequences play a role inincreasing mRNA stability. Higher levels of translation or proteinsynthesis were achieved by the inclusion of the Rbcs 5' untranslatedsequence containing a translational enhancer, thereby increasing thepool of protein precursors for importation into the plastidiccompartment. The level of Cab assembled into thylakoid membranes andLhcIIb complexes was further elevated by using a more efficient transitpeptide. Switching the type I LhcIIb Cab transit peptide with the onefrom the small subunit of ribulose-1,5-bisphosphate carboxylase enhancedthe level of import into the chloroplast. The increase in type I LhcIIbCab content inside the chloroplast allowed the LhcIIb antennae toincorporate the extra proteins and as a result increased the size of theantennae. Any transit peptide that will cause an increase in type ILhcIIb Cab in the chloroplast by replacing the Cab transit peptide canproduce in a similar elevating effect. It is also possible to achievelower levels of importation by using less efficient transit peptides andthus regulate the amount of protein expression. The experimentsdescribed herein show that the presence of the Rbcs transit peptideenhances importation of a wide range of plastid-destined proteinprecursors and probably represents the highest efficiency transitpeptide characterized to date.

The term "promoter" or "promoter region" refers to a sequence of DNA,usually upstream (5') to the coding region of a structural gene, whichcontrols the expression of the coding region by providing recognitionand binding sites for RNA polymerase and any other factors required fortranscription to start at the correct site.

There are generally two types of promoters, inducible and constitutivepromoters. The term "constitutive" as used herein does not necessarilyindicate that a gene is expressed at the same level in all cell types,but that the gene is expressed in a wide range of cell types, althoughsome variation in abundance is often detected.

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer the DNAsequences or genes will not be transcribed. Typically a protein factor(or factors), that binds specifically to an inducible promoter toactivate transcription, is present in an inactive form which is thendirectly or indirectly converted to an active form by the inducer. Theinducer can be a chemical agent such as a protein, metabolite, growthregulator, herbicide or phenolic compound, or a physiological stressimposed directly by heat, cold, salt, or toxic elements or indirectlythrough the action of a pathogen or disease agent such as a virus. Theinducer can also be an illumination agent such as light, darkness andlight's various aspects, which include wavelength, intensity, fluence,direction and duration. A plant cell containing an inducible promotermay be exposed to an inducer by externally applying the inducer to thecell or plant such as by spraying, watering, heating or similar methods.If it is desirable to activate the expression of a gene at a particulartime during plant development, the inducer can be applied at that time.

Examples of such inducible promoters include heat shock promoters, suchas the inducible hsp70 heat shock promoter of Drosphilia melanogaster(Freeling, M. et al. (1985) Ann. Rev. of Genetics 19:297-323); a coldinducible promoter, such as the cold inducible promoter from B. napus(White, T. C. et al. (1994 Plant Physiol. 106:917); and the alcoholdehydrogenase promoter which is induced by ethanol (Nagao, R. T. et al.,Miflin, B. J., Ed. Oxford Surveys of Plant Molecular and Cell Biology,Vol. 3, p 384-438, Oxford University Press, Oxford 1986).

Among sequences known to be useful in providing for constitutive geneexpression are regulatory regions associated with Agrobacterium genes,such as nopaline synthase (Nos), mannopine synthase (Mas) or octopinesynthase (Ocs), as well as regions regulating the expression of viralgenes such as the 35S and 19S regions of cauliflower mosaic virus (CaMV)(Brisson et al. (1984) Nature 310:511-514), or the coat promoter of TMV(Takamatsu et al. (1987) EMBO J. 6:307-311).

Other useful plant promoters include promoters which are highlyexpressed in phloem and vascular tissue of plants such as the glutaminesynthase promoter (Edwards et al. (1990) Proc. Natl. Acad. Sci. USA87:3459-3463), the maize sucrose synthetase 1 promoter (Yang et al.(1990) Proc. Natl. Acad. Sci. USA 87:4144-4148), the promoter from theRol-C gene of the TLDNA of Ri plasmid (Sagaya et al., Plant CellPhysiol., 3:649-653), and the phloem-specific region of the pRVC-S-3Apromoter (Aoyagi et al., Mol. Gen. Genet., 213:179-185 (1988)).Alternatively, plant promoters such as the small subunit of Rubisco(Rbcs) promoter (Coruzzi et al., EMBO J., 3:1671-1679 (1984); Broglie etal., Science, 224:838-843 (1984)), or heat shock promoters, e.g.,soybean HPS17.5-E or HPS17.3-B (Gurley et al. (1989) Mol. Cell. Biol.6:559-565 (1986)) may be used.

Other useful promoters which can be used according to the presentinvention include: the low temperature and ABA-responsive promoter Kin1,cor6.6 (Wang et al. (1995) Plant Mol. Biol. 28:605; Wang and Cutler(1995) Plant Mol. Biol. 28:619); the ABA inducible promoter from EM genewheat (Marcotte Jr. et al. (1989) Plant Cell 1:969); the phloem-specificsucrose synthase promoter, ASUS1, from Arabidopsis (Martin et al. (1993)Plant J. 4:367); the root and shoot promoter, ACS1 (Rodrigues-Pousada etal. (1993) Plant Cell 5:897); the seed-specific 22 kDa zein proteinpromoter from maize (Unger et al. (1993) Plant Cell 5:831); the ps1lectin promoter in pea (de Pater et al. (1993) Plant Cell 5:877); thephas promoter from Phaseolus vulgaris (Frisch et al. (1995) Plant J.7:503); the late embryo-abundant lea promoter (Thomas, T. L. (1993)Plant Cell 5:1401); the fruit-specific E8 gene promoter from tomato(Cordes et al. (1989) Plant Cell 1:1025); the meristematictissue-specific PCNA promoter (Kosugi et al. (1995) Plant J. 7:877); theNTP303 pollen-specific promoter (Weterings et al. (1995) Plant J. 8:55);the late embryogenesis stage-specific OSEM promoter (Hattori et al.(1995) Plant J. 7:913); the ADP-glucose pyrophosphorylasetissue-specific promoter for guard cells and tuber parenchyma cells(Muller-Rober et al. (1994) Plant Cell 6:601); the Myb conductivetissue-specific promoter (Wissenbach et al.(1993) Plant J. 4:411); andthe plastocyanin promoter from Arabidopsis (Vorst et al. (1993) Plant J.4:933).

The construct of the present invention also includes a 5' untranslatedleader sequence, which acts as a translational enhancer. Specificinitiation signals may be required for efficient translation of thecoding sequences. These signals include the ATG initiation codon andadjacent sequences. The initiation codon must be in phase with thereading frame of the coding sequence to ensure translation of thesequence. The translation control signals and initiation codon can be ofa variety of origins, both natural and synthetic. Translational controlsignals and initiation codon can be provided from the source of thetranscriptional initiation region, or from the structural gene. Thissequence can also be derived from the promoter selected to express thegene, and can be specifically modified so as to increase translation ofthe mRNA.

An example of a translational enhancer of the present invention is the5' untranslated region of the pea small subunit ofribulose-1,5-bisphosphate carboxylase. Other nucleic acid sequencesdemonstrating translational enhancing activity have been reported forleader or 5' untranslated sequences such as from the ferrodoxin-bindingprotein gene psaDb (Yamamoto et al. (1995) J. Biol Chem. 270:12466),ferredoxin (Dickey et al. (1994) Plant Cell 6:1171), the 68 base leaderfrom tobacco mosaic virus (TMV) (Gallie et al. (1987) Nucleic Acids Res.15:3257) and the 36 base leader from alfalfa mosaic virus (Jobling etal. (1987) Nature 325:622). These translational enhancers can be used inplace of the Rbcs translational enhancer signals in the presentinvention. Translational enhancing activity is most likely to be presentin the 5' untranslated nucleic acid sequence of most other genes andtheir corresponding transcripts and can vary in strength and efficiency(see review by Gallie. 1993 Ann. Rev. Plant Physiol. Plant Mol. Biol.44, 77). Such nucleic acid sequences, if demonstrated to containtranslational enhancing effects, can also be used in the presentinvention. A translational enhancer demonstrating appropriate levels ofenhancement can be selected to obtain a suitable level of translationalenhancement in the constructs of the invention.

The construct of the present invention also includes a transit peptide.A "transit peptide" refers to a peptide which is capable of directingintracellular transport of a protein joined thereto to a plastid in aplant host cell. The passenger protein may be homologous or heterologouswith respect to the transit peptide. Chloroplasts are the primaryplastids in photosynthetic tissues, although plant cells are likely tohave other kinds of plastids, including amyloplasts, chromoplasts, andleucoplasts. The transit peptide of the present invention is a transitpeptide which will provide intracellular transport to the chloroplastsas well as other types of plastids. In many cases, transit peptides canalso contain further information for intraorganellar targeting withinthe plastid to sites of function such as outer and inner envelopemembranes, stroma, thylakoid membrane or thylakoid lumen. Depending onthe source of the transit peptide, the precursor proteins may displaydifferences in import behavior and import activity such as efficiency.These differences in import behavior are not attributed solely to thefunction of the transit peptide but also to the passenger protein andare most likely due to interactions between the two portions (Ko and Ko,1992 J. Biol. Chem. 267, 13910). In photosynthetic prokaryotes, such asthe cyanobacteria, proteins can be targeted to the photosynthetic andplasma membranes or to biochemical pathways involving the reduction ofsugars and formation of photosynthetic products.

The transit peptide for the constituent polypeptide of thelight-harvesting chlorophyll a/b-protein complex is rich in serine,especially near the NH₂ -terminus, where 7 of the first 13 residues areserine. An abundance of serine also occurs near the NH₂ -terminus of thetransit peptide for the small subunit of Rbc from pea (Cashmore, A. R.,Genetic Engineering of Plants, Eds. Kosuge, T. et al. (Plenum Press, NewYork, pp. 29-38 (1983)), soybean (Berry-Lowe, S. L. et al. (1982) J.Mol. Appl. Genet. 1:483-498), and Chlamydomonas (Schmidt, G. W. et al.(1989) J. Cell Biol. 83:615-623). Both the transit peptides for thelight-harvesting chlorophyll a/b-protein complex and for the smallsubunit of Rbc function in the specific translocation of polypeptidesacross the chloroplast envelope. However, the final destination of thesepolypeptides is quite distinct, with the light-harvesting chlorophylla/b-protein complex residing as integral membrane proteins in thechloroplast thylakoid and the small subunit of Rbc residing as acomponent of a soluble protein in the chloroplast stroma.

In one embodiment, the transit peptide is from the small subunit of Rbc.The level of Cab assembled into the thylakoid membrane and the LhcIIbcomplex was further elevated by using a more efficient, heterologoustransit peptide. The switching of the type I LhcIIb Cab transit peptidewith the one from the small subunit of ribulose-1,5-bisphosphatecarboxylase enhanced the level of Cab import into the chloroplast. Theincrease in type I LhcIIb Cab content inside the chloroplast allowed theLhcIIb antennae to incorporate the extra proteins and as a resultincreased the size of the antennae.

The gene encoding the protein to be transcribed and incorporated into aplastid or cell of a photosynthetic organism is not particularlylimited. Those of skill in the art will recognize that other genesencoding pigments (such as the phycobiliproteins) or pigment-bindingproteins (such as carotenoid-binding proteins) could be utilized toenhance the efficiency of the light-harvesting reactions. Many processesof photosynthesis could be similarly enhanced. For example, genesencoding the subunits of ATP synthase and ferredoxin involved inelectron transport could be incorporated into the constructs of thisinvention to enhance electron transport. Alternatively, the expressionand import of pyruvate kinase, acetyl-CoA carboxylase and acyl carrierproteins could be increased, thus amplifying a biosynthetic pathway,such as carbon/lipid metabolism.

Any gene encoding a chlorophyll a/b binding (Cab) protein can beselected as a structural gene. The chlorophyll a/b binding proteinsinclude LhcI of four different types, LhcII of types I to III, CP29,CP26, CP24 and early light-induced proteins (Green B. R. (1991) TrendsBiochem. Sci. 16:181-186). These include genes or cDNAs encodingchlorophyll a/b binding proteins that can belong to the complexesLhcIIa, LhcIIb, LhcIIc, LhcIId, LhcIIe and any other uncharacterizedsubcomplexes of LhcII. The same gene construction scheme can be appliedas well to genes or cDNAs encoding chlorophyll a/b binding proteins ofLhcI which include chlorophyll a/b binding proteins of LhcIa, LhcIb, andLhcIc of photosystem I.

LhcII is the major complex comprising the most abundant members of thefamily of chlorophyll a/b binding proteins, accounting for approximately50% of total chlorophyll in the biosphere, and for the most chlorophyllb in green plants. Thus, a gene encoding a LhcII chlorophyll a/b bindingprotein would be a preferred gene for targeting to increase the amountof chlorophyll a/b binding proteins.

In all plant species examined to date, chlorophyll a/b binding proteinsof LhcII are encoded by a multi-gene family, comprising at least fivegenes in Arabidopsis, six genes in Nicotiana tabacum, eight genes in N.plumbaginifolia, and up to 15 genes in tomato (Jansson, S. et al. (1992)Plant Mol. Biol. Rep. 10:242-253). Thus, any of these genes would be asuitable target for increasing the amount of chlorophyll a/b bindingprotein. Table 1 provides a more complete list of genes encodingchlorophyll a/b binding proteins, including those presently in thenucleic acid sequence data banks such as that represented and listed inTable 2 of the publication by Jansson, et al. (1992) supra.

                                      TABLE 1                                     __________________________________________________________________________    Genes encoding chlorophyll a/b binding proteins, and their relation to         designations for chlorophyll-protein complexes                                  Gene Product/Pigment-Protein Complex                                          Green et al. 1991                                                             Trends Biochem Thornber Bassi                                                Gene Sci. 16, 181 et al. 1991 et al. 1990 References (genes) References                                    (proteins)                                     __________________________________________________________________________    Lhca                                                                             Type I LhcI                                                                           Lhc Ib                                                                             LhcI-730                                                                           Hoffman et al., 1987                                                                    Ikeuchi et al., 1991                             1    Jansson & Gustafsson Knoetzel et al., 1992                                   1991 Palomares et al.,                                                        1991                                                                      Lhca Type II LhcI Lhc Ia LhcI-680 Stayton et al., 1987 Ikeuchi et al.,                                     1991                                             2    Pichersky et al., 1988 Knoetzel et al., 1992                                 Jansson & Gustafsson                                                          1991                                                                      Lhca Type III LhcI Lhc Ia LhcI-680 Pichersky et al., 1989 Ikeuchi et                                       al., 1991                                        3    Jansson & Gustafsson Knoetzel et al. 1992                                    1991                                                                      Lhca Type IV LhcI Lhc Ib LhcI-730 Schwartz et al., 1991a Ikeuchi et                                        al., 1991                                        4    Zhang et al., 1991 Schwartz et al., 1991a                                     Knoetzel et al. 1992                                                     Lhcb Type I LhcII Lhc IIb 28 LhcII Chitnis & Thornber, Jansson et al.,                                     1990                                             1  kDa  1988* Green et al., 1992                                              Lhcb Type II LhcII Lhc IIb 27 LhcII Chitnis & Thornber, Jansson et al.,                                    1990                                             2  kDa  1988* Green et al., 1992                                              Lhcb Type III LhcII Lhc IIb 25 LhcIIa Schwartz et al., 1991b Bassi &                                       Dainese, 1990                                    3  kDa  Brandt et al., 1992 Morishige &                                            Thornber, 1991                                                                Bassi & Dainese, 1992                                                         Green et al., 1992                                                       Lhcb Type II CP29 Lhc IIa CP29 Morishige & Henrysson et al., 1989                                           4    Thornber, 1992 Pichersky et al.,                                        1991                                                  Morishige &                                                                   Thornber, 1992                                                           Lhcb Type I CP29 Lhc IIc CP24 Pichersky et al., 1991 Pichersky et al.,                                     1991                                             5    Sorenson et al., 1992 Morishige &                                             Thornber, 1992                                                           Lhcb CP24 Lhc IId LhcI-730 Schwartz & Pichersky, Morishige et al., 1990       6    1990 Spangfort et al., 1990                                            __________________________________________________________________________     *Because numerous genes of these kinds have been cloned and sequenced, a      review article is given as reference.                                         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Biol. 12, 257.                              Schwartz et al. 1991a, FEBS Lett. 280, 229.                                   Zhang et al. 1991, Plant Physiol 96, 1387.                                    Chitnis and Thornber, 1988, Plant Mol. Biol. 11, 95.                          Jansson et al. 1990, Biochim. Biophys. Acta. 1019, 110.                       Green et al. 1992, FEBS Lett. 305, 18.                                        Schwartz et al. 1991b, Plant Mol. Biol. 17, 923.                              Brandt et al. 1992, Plant Mol. Biol. 19, 699.                                 Bassi & Dainese, 1990, In: Current Research in Photosynthesis. Vol II,        Baltscheffsky, M. (ed.) pp 209-216.                                           Morishige & Thornber, 1991, FEBS Lett. 293:183.                               Bassi & Dainese, 1992, In: Regulation of chloroplast biogenesis.              ArgyroudiAkoyonoglou, J. (ed.) pp. 511.520.                                   Morishige & Thornber, 1992, Plant Physiol. 98, 238.                           Henrysson et al. 1989, Biochim. Biophys. Acta. 977, 301.                      Pichersky et al. 1991., Mol. Gen. Genet. 227, 277.                            Sorensen et al. 1992, Plant Physiol. 98, 1538.                                Schwartz & Pichersky, 1990. Plant Mol. Biol. 15, 157                          Morishige et al. 1990. FEBS Lett. 264, 239                                    Spangfort et al. 1990. In: Current Research in Photosynthesis. Vol II,        Baltscheffsky, M. (ed.) pp 253-256.                                      

The Cab protein of PSII encoded by ICABPSII is the major lightharvesting antenna associated with PSII and contains 40-60% of the totalchlorophyll in the mature chloroplast (Boardman et al. (1978) CurrentTopics in Bioenergetics, 8:35-109). Further, within PSII, there is avery high sequence homology between type I and type II Cab proteins(Pichersky et al. (1989) Plant Mol. Biol. 12:257). Thus, targeting thisgene will significantly alter the chlorophyll content.

Useful genes encoding a chlorophyll a/b binding protein, which can beused according to the present invention, include:

a) the light harvesting complex I--complexes of photosystem I, such asLhcal type I, the major Cab proteins of PSI, e.g. Lhca1*1 (Hoffman etal. (1987) Proc. Natl. Acad. Sci. USA 84:8844); Lhca2; Lhca3 type III,the major Cab proteins of PSI, e.g. Lhca3*1 (Pichersky et al. (1989)Plant Mol. Biol. 12:257); Lhca4; and

b) the light harvesting complex II--complexes of photosystem II, such asLhcb1; Lhcb2 type II, the major Cab proteins, e.g. Lhcb2*1 (Pichersky etal. (1987) Plant Mol. Biol. 9:109); Lhcb3 type III, the major Cabproteins, e.g. Lhcb3*1 (Schwartz et al. (1991) FEBS Lett. 280:229);Lhcb4; Lhcb5; and Lhcb6.

In one embodiment of the present invention, a nuclear gene encoding aconstituent polypeptide of the light-harvesting chlorophyll a/b proteincomplex (type I Lhc IIb), which has been isolated from pea (Pisumsativum) (Cashmore, A. R. (1984) Proc. Natl. Acad. Sci. USA81:2960-2964) was used. In other embodiments, the cab genes selected canrepresent the remaining two proteins of LhcIIb or other major proteinsof LhcIb. In addition to type I, there are two other Cab proteins ofLhcIIb: type II and type III. Since these two proteins are constituentsof the major light harvesting complex LhcIIb, they may also play animportant role in low light and may also be preferred. The LhcIb complexis the major complex for photosystem I and consists of two Cab proteins,type I and III LhcIb Cab.

The construct of the present invention further comprises a 3'untranslated region. A 3' untranslated region refers to that portion ofa gene comprising a DNA segment that includes a polyadenylation signaland can include other regulatory signals capable of affecting mRNAprocessing, mRNA stability, or gene expression. The polyadenylationsignal is usually characterized by effecting the addition of apolyadenylic acid tract to the 3' end of the mRNA precursor.Polyadenylation signals are commonly recognized by the presence ofhomology to the canonical form 5' AATAAA-3' although variations are notuncommon.

Examples of suitable 3' regions are the 3' transcribed non-translatedregions containing a polyadenylation signal of Agrobacterium tumorinducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene)and plant genes such as the soybean storage protein genes and the genefor the small subunit of ribulose-1,5-bisphosphate carboxylase. Othersuitable 3' sequences may be derived from any characterized gene fromplants as well as from other organisms such as animals if they aredeemed appropriately functional in the environment of a transgenic plantcell or cell of a photosynthetic organism. In one embodiment of theinvention, the 3' untranslated region is derived from the structuralgene of the present construct.

When specific sequences are referred to in the present invention, it isunderstood that these sequences include within their scope, sequenceswhich are "substantially similar" to the specific sequences. Sequencesare "substantially similar" when at least about 80%, preferably at leastabout 90% and most preferably at least about 95% of the nucleotidesmatch over a defined length of the molecule. Sequences that are"substantially similar" include any sequence which is altered to containa substitution, deletion, or addition of nucleotides compared to thesequences of this invention, especially substitutions which rely on thedegeneracy of the genetic code, and which have similar characteristics(i.e., function). DNA sequences that are substantially similar can beidentified and isolated by hybridization under high or moderatestringency conditions, for example, which are chosen so as to not permitthe hybridization of nucleic acids having non-complementary sequences."Stringency conditions" for hybridization is a term of art which refersto the conditions of temperature and buffer concentration which permithybridization of a particular nucleic acid to another nucleic acid inwhich the first nucleic acid may be perfectly complementary to thesecond, or the first and second may share some degree of complementaritywhich is less than perfect. "High stringency conditions" and "moderatestringency conditions" for nucleic acid hybridizations are explained onpages 2.10.1-2.10.16 and pages 6.3.1-6 in Current Protocols in MolecularBiology (Ausubel, F. M. et al., eds., Vol.1, containing supplements upthrough Supplement 29, 1995), the teachings of which are herebyincorporated by reference.

To aid in identification of transformed plant cells, the constructs ofthis invention may be further manipulated to include genes coding forplant selectable markers. Useful selectable markers include enzymeswhich provide for resistance to an antibiotic such as gentamycin,hygromycin, kanamycin, or the like. Similarly, enzymes providing forproduction of a compound identifiable by color change such as GUS(β-glucuronidase), or by luminescence, such as luciferase, are useful.

The constructs of the present invention can be introduced into plantcells through infection with viruses or bacteria or direct introductionby physical or chemical means. Examples of indirect (infection) anddirect methods include Ti plasmids, Ri plasmids, plant virus vectors,micro-injection, microprojectiles, electroporation, and the like. Forreviews of such techniques see, e.g., Weissbach and Weissbach, Methodsfor Plant Molecular Biology, Academic Press, New York, Section VIII, pp.421-463 (1988); and Grierson and Corey, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9 (1988). The term "transformation" as usedherein, refers to the insertion of a construct into a plant cell or thecell of a photosynthetic organism by any of the above methods.

Methods of regenerating whole plants from plant cells are known in theart (See, e.g., Plant Molecular Biology Manual, (Eds. S. B. Gelvin, R.A. Schilperoort) Kluwer Acad. Publishers (1988)), and the method ofobtaining transformed and regenerated plants is not critical to thisinvention. In general, transformed plant cells are cultured in anappropriate medium, which may contain selective agents such asantibiotics, where selectable markers are used to facilitateidentification of transformed plant cells. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be used toestablish repetitive generations, either from seeds or using vegetativepropagation techniques.

Also considered part of this invention are plants and otherphotosynthetic organisms containing the nucleic acid construct of thisinvention. Suitable plants include both monocotyledons and dicotyledons,as well as gymnosperms and lower plants (e.g., ferns, bryophytes)included in the kingdom Plantae, and lichens. Examples of preferredmonocotyledons include rice, corn, wheat, rye and sorghum. Examples ofpreferred dicotyledons include canola, pea, soybeans, sunflower,tobacco, cotton, sugar beet, petunia, tomato, broccoli, lettuce, apple,plum, orange, lemon, and rose. Other photosynthetic organisms can alsobe used as hosts for the construct of the present invention. Theseinclude the single-celled eukaryotic and multicellular algae, such asPorphyra sp., Chondrus crispus, Gigartina sp., Eucheuma sp., Laminariasp., Macrocystis sp., Nereocystis leutkeana, Chlamydomonas reinhardtii,chlamydomonas moewusii, Euglena gracilis, Cryptomonas Φ, and Ochromonassinensis. This invention also includes prokaryotes which lack plastidsbut have a photosynthetic apparatus, such as the cyanobacteria(blue-green algae) and photosynthetic microbes, which include, forexample, Anacystis nidulans, Spirulina sp., Synechococcus sp.,Rhodobacter sphaeroides, Rhodobacter capsulatus, Chloroflexusaurantiacus, and Heliobacterium chlorum. Those of skill in the art canrecognize the examples given above are not limiting.

Transgenic plants can be used to provide plant parts according to theinvention for regeneration or tissue culture of cells or tissuescontaining the constructs described herein. Plant parts for thesepurposes can include leaves, stems, roots, flowers, tissues, epicotyl,meristems, hypocotyls, cotyledons, pollen, ovaries, cells, andprotoplasts, or any other portion of the plant which can be used toregenerate additional transgenic plants, cells, protoplasts or tissueculture.

Seed of transgenic plants are provided by this invention and can be usedto propagate more plants containing the constructs of this invention.These descendants are intended to be included in the scope of thisinvention if they contain the constructs of this invention, whether ornot these plants are selfed or crossed with different varieties ofplants.

The constructs of this invention provide materials and methods throughwhich genetic improvements in crop cultivars can be made to produce asubstantial enhancement in productivity and the economic value of crops.Most of the experimental strategies in the agribiotechnology andagricultural industries are aimed at enhancing the productivity of cropplants to maximize the returns per unit of farmland. Although thepreferred approach is to increase yield directly, productivity can alsobe enhanced by indirect means, such as reduction of input cost (e.g.,fertilizers and water) or by reducing losses due to disease, insects andcompetition. These indirect results can be effected by incorporatingnovel traits into crop plants that will lower the need for fertilizers,confer disease resistance, repel damaging insects or sustain herbicides.Increases in productivity can also result from improving theadaptability of the plant to other unfavorable environmental conditions.Further increases can be achieved by combinations of these traits,through the use of molecular procedures and making hybrids.

Most molecular attempts to alter photosynthesis, both direct andindirect, have resulted in the inhibition of photosynthesis. Thesestudies are reviewed by Furbank and Taylor (1995) Plant Cell 7:797 andStitt and Sonnewald (1995) Ann. Rev. Plant Physiol. Plant Mol. Biol.46:341. The methods used primarily involved reduction, via antisensetransgenes, of enzymes involved in photosynthetically-related processes.Ko et al. (1992 Research in Photosynthesis Vol III: Proceedings of theIXth International Congress on Photosynthesis, Ed. N. Murata.

Kluwer Academic Press, pp. 445-448) demonstrated a positive functionalchange to tobacco.

The organizational diversity of the light-harvesting complexes and theCab proteins involved suggests that variations in molecularrelationships between different light harvesting complexes/proteins isone of the key mechanisms of the plant's adaptability to changing lightconditions. For instance, a possible reorganizational event to causeadaptation to low lighting conditions could simply be the enlargement ofthe light harvesting complexes to gather more light by virtue ofantennae size or surface area. Larger antennae would capture more lightfor conversion to chemical energy. Therefore enhancement of the plant'sflexibility to reorganize the light harvesting machinery in response tovarying light conditions can benefit the plant. Limitations onflexibility can be due to limiting levels of functional Cab proteinsexpressed in the plants; therefore elevations in the levels of Cabproteins will relieve the limitations. Relieving these molecularlimitations can result in significant changes to photosynthesis andinterrelated activities and processes, giving rise to changes toproductivity and yield and improvements in the marketability and valueof plant and other products from crop plants. For instance, geneticmodifications aimed at enhancing photosynthesis are especially importantin situations where crop production must be profitable despite thelimitations imposed by diverse environmental conditions, e.g., limitinglight conditions. Enhancing photosynthesis and related activities canalso have a significant impact on crops engineered to produce non-plantproducts, e.g., health products, by providing the energy to drive theproduction of such products. The implications of this type of directedgenetic modification approach are diverse and cannot be listedindividually or estimated as a single economic benefit. Impact of thiswork can be significant, from improved crop productivity to indirectsavings due to reduction in the lighting requirements of greenhousegrown plants. The technology applies not only to crop plants but also tohorticultural plants, from house plants to orchids to ornamentals. Dueto the universality of the photosynthetic process being enhanced, thetechnology is most likely to be beneficial and applicable to allphotosynthetic organisms and plant varieties. In addition to theadvancement of knowledge of photosynthesis and related activities, thereare four principal categories of benefits to agriculture andhorticulture provided by this invention:

1) Improved marketability of plant products (e.g., greener plants);

2) Improved productivity under low light conditions;

3) Improved planting density; and

4) Improved yields.

The development of technologies for the transfer of genes into plantcells and regeneration of intact and fertile plants from the transformedcells provides methods to modify certain of these molecular parametersto provide flexibility for the enhancement of a plant's photosyntheticcapacity in low light. Overproduction and elevation of functional Cabproteins of the light harvesting antennae of photosystem II enable aplant to reorganize and harvest more light for photosynthesis.Modifications which cause a positive effect on photosynthesis can giverise to new desirable traits that have widespread benefits inagriculture and horticulture. These novel traits in the form ofgenetically engineered plants can provide plants with advantages in thefield, greenhouse or any other form of growing practice compared withtheir normal unaltered counterparts. Advantageous traits can also beintroduced through traditional breeding strategies to provide anydesirable recombinant plant lines, e.g., elite lines, with thebeneficial novel traits in addition to established desirableagronomically important phenotypes.

In particular, enhancing chlorophyll binding proteins in plastids canproduce a higher chlorophyll content in plastids. Greener plants havecommercial value in horticulture, both in the production of pottedplants for house and garden, and for landscaping purposes. The improvedcolor and growth resulting from incorporation of the constructs of thisinvention can provide superior phenotypes in all varieties of plants,including turf grasses, ground covers, flowers, vegetables, trees andshrubs. Further, elevated chlorophyll levels will produce post-harvestcolor retention for fresh produce or dried plant products. Increasedpigments levels of carotenoids and phycobiliproteins can also havecommercial value for the same purposes. Further, increased levels ofcarotenoids can lead to increased nutritive value in foods such ascarrots, and can increase resistance of plants to the damaging effectsof ultraviolet light.

The transgenic plants of this invention demonstrate many other improvedproperties. Transgenic plants are bigger than their wild-typecounterparts, even under high light conditions. Following inclusion of agene encoding the Cab binding protein, they are greener and demonstratemore robust growth than wild-type plants. The constructs of thisinvention can also provide plants with a means to withstandtransplantation shock. Transplanted transgenic plants recover from thesetbacks of transplanting more rapidly than wild-type plants.

Seeds of transgenic plants are larger and germinate more rapidly thanseeds produced by wild-type plants, forming more robust seedlings.Faster germination results in larger shoots and extensive roots whichare less susceptible to the fungal and bacterial pathogens that attackgerminating seeds and seedlings. Further, seedlings which quicklyestablish extensive and deep root systems are more resistant to droughtstress. Thus, transgenic seeds and seedlings are more commerciallyvaluable than naturally-occurring varieties.

Further, the constructs and methods of this invention can be used toenhance stem girth, thereby enhancing support of a plant. This isespecially valuable for fruit bearing crops such as tomatoes, pears,apples, oranges, lemons, and the like. Larger and sturdier stems willpermit the development of varieties that can bear and support morefruit. Further, newly-transplanted ornamental plants, including treesand small shrubs subject to wind can benefit from enhanced stem girthfor support until they establish strong root systems.

The growth benefits afforded to transgenic plants and plant cells ofthis invention can be reproduced by incorporating the constructs of thisinvention into single-celled photosynthetic organisms and plant tissueculture. Thus, more rapid production of plant products which are noteasily synthesized, such as taxol and other cell wall products, whichare produced in slow-growing plants and in tissue culture, can berealized. Further, increased photosynthesis and the subsequent increasein growth under low light intensities means that plant regeneration canbe accelerated and illumination can be reduced for tissue culture andplant production.

In fact, the reduced light requirement of plants described in thisinvention can permit these plants to be grown at lower cost in low lightfacilities such as caves (currently used in the floral industry) andunder denser canopies. More highly developed technological uses includechambers associated with life support systems for space travel. Spaceagencies would like to enhance the growth of photosynthetic organisms,and lower light requirements of such organisms would make such a systemeasier and less expensive to manufacture and operate.

One especially useful embodiment of this invention is the production ofshade-tolerant varieties of grasses. These varieties can be plantedwhere present varieties will not grow due to reduced illuminationlevels. This includes, for example, portions of lawns shaded by trees,as well as indoor stadiums where astroturf is now required because oflight limitations. NFL football teams are currently converting outdoorstadium fields from Astroturf to living grass due to Astroturf-relatedinjuries. Indoor stadium fields are under light-limiting domes, however,and grass cannot be grown in these fields unless a more shade-tolerantvariety is provided.

In another aspect of this invention, the constructs of this inventioncan be used to produce plants with less variability in their growthpattern. Transgenic plants provided by this invention grow more evenlythan wild-type plants under both greenhouse and field conditions becausethe available light is used effectively by all parts of the plants. Thischaracteristic can produce yield advantages in commercially-grown plantssuch as corn or soybeans, where the lower shaded leaves can grow morevigorously, producing an increased biomass which not only contributes toseed yield but also shades out weeds.

The DNA construct provided by this invention can be used as a planttransformation marker, based on differences in coloration, shade/lowlight responses and faster growth and/or development, especially underlow light conditions. The use of naturally-occurring plant DNA sequencesallows the detection of integration of exogenous DNA constructs inphotosynthetic cells and organisms without the regulatory problemsassociated with foreign selectable markers. In particular, there isprovided a method for detecting transformation in plants, plant tissueor a photosynthetic organism consisting of: preparing a DNA constructcomprising a promoter region, a 5' untranslated region containing atranslational enhancer, a plastid-specific transit peptide, a geneencoding a plastid protein the expression of which is detectable, and a3' untranslated region containing a functional polyadenylation signal;inserting the DNA construct into a cloning vector; and transforming aplant, tissue culture or photosynthetic organism with the cloning vectorso that the protein is expressed, wherein expression of the protein isindicative of transformation. Preferably, expression and import of theprotein into plastids or to the photosynthetic apparatus of cells areincreased relative to wild-type plastids and cells. In one embodiment,the encoded protein is chlorophyll a/b binding protein.

The marker gene which is expressed can provide a visibly reactiveresponse, i.e., cause a distinctive appearance or growth patternrelative to plants or cells of photosynthetic organisms not expressingthe selectable marker gene, allowing them to be distinguished from otherplants, parts of plants, and cells of photosynthetic organisms forpurposes of identification. Such a characteristic phenotype (e.g.,greener cells) allows the identification of protoplasts, cells, cellgroups, tissues, organs, plant parts or whole plants containing theconstructs. Green pigmentation in cells can be easily measured andscreened by using techniques such as FACS (fluorescence-activated cellsorting). Galbraith, D. W. (1990) Methods Cell Biol. 33:547-547. Ifanother gene has been incorporated with the construct, detection of themarker phenotype makes possible the selection of cells having a secondgene to which the marker gene has been linked. This second genetypically comprises a desirable phenotype which is not readilyidentifiable in transformed cells, but which is present when the plantcell or derivative thereof is grown to maturity, even under conditionswherein the selectable marker phenotype itself is not apparent.

The following examples describe specific aspects of the invention todepict the invention and provide a description of the methods used toprovide the constructs of the invention and to identify their functionin organisms. The examples should not be construed as limiting theinvention in any way.

EXEMPLIFICATION Example 1

In vitro Translation and Protein Import Analysis

A variety of different gene fusions were prepared which demonstrate thatthe Rbcs 5' untranslated region (5'UTR) and the Rbcs transit peptideconfer higher levels of translation and importation, respectively, ofchimeric gene constructs. These in vitro import assay and translationdata are summarized in Table 2. In many cases, the Rbcs transit peptideconferred higher levels of import for the passenger protein into thechloroplast. The translation-enhancing effect of the 5'URT of the Rbcsgene was demonstrated in an in vitro wheat germ translation system.These data show the Rbcs 5'URT is a translational enhancer bydefinition.

The analysis of protein importation and related aspects, such asefficiency, was carried out using in vitro radiolabelled proteins and invitro import assays. Radiolabelled proteins were synthesized fromcorresponding DNA templates via transcription and are depicted in Table2. All transcription plasmids depicted were propagated in theEscherichia coli strains HB101 or the JM101-109 strain series. Thetransformation of various bacterial strains was carried out usingstandard protocols (see, e.g., Molecular Cloning: A Laboratory Manual,Sambrook et al. 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). Plasmid DNA was isolated from the bacterial strains harboring thecorresponding plasmids using standard protocols (Sambrook et al. 1989,supra). A variety of transcription plasmids containing different fusions(Table 2) were linearized at appropriate restriction sites 3' to thegene fusion DNA template. Restriction enzyme digestion buffers anddigestion conditions were employed according to the protocols providedby the supplier of each particular enzyme. The gene fusion templates canbe created, inserted and propagated in a variety of commerciallyavailable transcription plasmids such as the pBLUESCRIPT series(Stratagene), the pBS series (Stratagene), the pGEM and pSP series(Promega) and pT7/T3 series (Pharmacia). Transcription plasmids usuallycontain multiple cloning regions for cloning manipulations and at leastone of the viral RNA polymerase promoters. These promoters can be, forexample, T7, T3 or SP6, which use the corresponding RNA polymerase.

Restriction enzyme was added to give 5-10 units per microgram of DNA andthe reaction mixture was adjusted to the appropriate final volume withwater. The final volumes were usually 200 μl and contained 10 μg ofplasmid DNA. Digestions were thoroughly mixed and carried out for 1-3 hat the appropriate suggested temperature. Digested templates werere-purified by phenol and chloroform:isoamyl alcohol extraction,centrifugation (usually in a microfuge) and the aqueous layer containingthe digested DNA concentrated by precipitation in two volumes of 100%ethanol in the presence of 0.3 M sodium acetate, pH 7.0. The phenol usedwas saturated with 0.1 M Tris-HCl pH 8.0 plus 0.1% (w/v) hydroxquinolineprior to use. The chloroform:isoamyl alcohol consisted of 24 volumes ofchloroform and 1 volume of isoamyl alcohol. Equal volumes of aqueousreaction mixture and phenol or chloroform:isoamyl alcohol were used ineach of the organic solvent extraction steps. The DNA precipitates werecollected by centrifugation, washed once with 70% (v/v) ethanol, driedand redissolved in a volume of 20 μl water prior to in vitrotranscription.

The templates were transcribed in vitro using the appropriate RNApolymerase corresponding to the promoter type according to Melton et al.(1984) Nucl. Acids Res. 12:7035. An unmethylated cap analog (GpppG),usually at a concentration of 0.5 mM (Pharmacia), was included in thereactions. A typical transcription reaction (200 μl total volume)consisted of DNA template (5 μg), RNA polymerase (40 units of eitherSP6, T3 or T7), transcription reaction buffer (final concentration of 40mM Tris-HCl pH 7.5, 6 mM MgCl₂, 2 mM spermidine, 10 mM NaCl),dithiothreitol (DTT, 5 mM), 40 units RNasin, and 0.25 mM of ATP, GTP,CTP and UTP. The transcripts were re-purified by phenol andchloroform:isoamyl alcohol extraction, centrifugation and the aqueouslayer containing the RNA transcripts concentrated by precipitation with750 μl of 100% ethanol in the presence of 0.3 M potassium acetate pH7.0. The transcripts (330 μl of the reaction) were concentrated afterprecipitation in ethanol by centrifugation, washed with 70% (v/v)ethanol/water, dried and redissolved in 34 μl of water in the presenceof 20-40 units of RNasin. Alternatively the transcript precipitates werestored at -70° C. until use.

The transcripts were then translated in a wheat germ extract systemcontaining either ³⁵ S-radiolabelled methionine (New England Nuclear orAmersham) or TRAN³⁵ S-Label (ICN) which contains both radiolabelledmethionine and cysteine. Wheat germ extracts were prepared according toErickson and Blobel (1983) Methods in Enzymol. 96:38 with somemodifications. The wheat germ extract was prepared by grinding 3 g ofuntoasted wheat germ (General Mills, Inc., Minneapolis, Minn.) using amortar and pestle in the presence of liquid nitrogen. Grinding proceededuntil a fine powder was achieved. The powdered wheat germ was thentransferred to a second prechilled mortar where grinding continued inthe presence of 12 ml homogenization buffer (100 mM potassium acetate,40 mM Hepes-KOH pH 7.6, 4 mM DTT, 2 mM CaCl₂, 1 mM magnesium acetate)until the mixture had a thick paste consistency. The homogenate was thentransferred to 30 ml Corex tubes and centrifuged for min at 31,000×g at4° C. The supernatant was recovered and centrifuged in a 15 ml Corextube for another 10 min at the same g force. The final supernatant wascarefully removed and its volume determined (typically 8-9 ml). Thesupernatant was loaded onto a Sephadex G-25 Fine (Pharmacia) column(2.5×20 cm) which was pre-sterilized by autoclaving. The columnconsisted of 20 g (giving a column bed of 2.5×18 cm) of Sephadex G-25Fine (Pharmacia, Sigma) presoaked overnight with chilled, sterilizedwater and equilibrated with column buffer (100 mM potassium acetate, 40mM Hepes-KOH pH 7.6, 4 mM DTT, 5 mM magnesium acetate) before use. Thewheat germ extract was eluted at a rate of 1-1.5 ml/min with columnbuffer and fractions were collected when the leading brown band migratedtwo-thirds down the column. The protein content of each fraction wasmonitored by measuring absorbance at 280 nm in a spectrophotometer. Thefractions encompassing the first peak were pooled and mixed. Aliquots ofthis first eluted peak were quick-frozen in liquid nitrogen and storedat -70° C. until use. A typical translation reaction consisted oftranscript from 330 μl of ethanol precipitate dissolved in 34 μl water,20-40 units of RNasin, 5-6 mM ATP, 0.4 mM GTP, 64 mM creatine phosphate,0.08-0.09 mM of each amino acid, except that either methionine ormethionine plus cysteine was not added, depending on the type of ³⁵ Slabelled amino acid(s) used, 4-8 units creatine phosphokinase,compensation buffer (final concentration: 100 mM potassium acetate pH7.0, 2 mM DTT, 0.3 mM magnesium acetate, 0.8 mM spermidine) and wheatgerm extract (80 μl in a 200 μl reaction). The amount of radiolabelledamino acid used in the translations was based on radiolabelledmethionine alone in the amount of 200 μCi per reaction. The translationreactions were carried out for 1.5 h at 26-27° C. and the translationproducts normally analyzed prior to import assays or other relatedexperimentation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) andfluorography. The amount of radiolabel incorporation was determined bycounting TCA-precipitable radioactive counts of 1 μl samples of eachtranslation reaction.

The intact chloroplasts used in the import assays were purified from peaseedlings (cv. Improved Laxton's Progress) as described by Bartlett etal. (1982) Methods in Chloroplast Molecular Biology, (eds. Edelman etal.) pp. 1081-1091 or Cline et al. (1985) J. Biol. Chem. 260:3691). Thegrowth conditions were identical to those described previously (Ko andCashmore (1989) EMBO J. 8:3187). Pea seedlings from 200 g of seeds weregrown for 9-11 days in growth chambers set at 21° C. under fluorescentlighting with 16:8 h light:dark photoperiod. Pea seedlings wereharvested and homogenized in cold grinding buffer (50 mM Hepes-KOH pH7.6, 0.33 M sorbitol, 0.05% (w/v) bovine serum albumin (BSA), 0.1% (w/v)ascorbate, 1 mM MgCl₂, 1 mM MnCl₂, 2 mM Na₂ EDTA) for 2-3 briefblendings of 5-10 sec at a setting of 5-6 on a Polytron Homogenizer. Allsteps were conducted at approximately 4° C. The homogenate was thenfiltered through three layers of Miracloth and the crude chloroplastscollected by centrifugation at 2,800×g for 3 min at 4° C. The crudechloroplast pellet was resuspended in 4 ml of grinding buffer andlayered onto a 10-80% Percoll gradient (50 mM Hepes-KOH pH 7.6, 0.33 Msorbitol, 0.05% (w/v) BSA, 0.1% (w/v) ascorbate, 0.15% (w/v)polyethylene glycol, 0.05% (w/v) Ficoll, 0.02% (w/v) glutathione, 1 mMMgCl₂, 1 mM MnCl₂, 2 mM Na₂ EDTA and Percoll). The gradients werecentrifuged in a swing out rotor at 10,000×g for 10 min at 4° C. and theintact chloroplast band near the bottom of the gradient was collectedand diluted at least five fold with 1× HS buffer (50 mM Hepes-KOH pH8.0, 0.33 M sorbitol). The intact plastids were collected bycentrifugation at 4,350×g for 2 min. This step was repeated with thepelleted chloroplasts by resuspending in 1× HS. The final pellet wasresuspended in 5 ml of 1× HS and an aliquot subjected to chlorophyllanalysis. Chlorophyll assays were performed as described by Arnon (1949)Plant Physiol. 24:1. Samples were extracted with 80% (v/v) acetone/20%water. Insoluble material was removed by centrifugation in a microfugefor 1 min at high speed. The supernatant was removed forspectrophotometric analysis of chlorophyll according to the Arnonconversion equation.

The in vitro import assays were performed in 0.3 ml volumes as describedby Bartlett et al. (1982) Methods in Chloroplast Molecular Biology, eds.Edelman et al., pp. 1081-1091. The reactions typically contained anequivalent of 100 μg chlorophyll of chloroplasts; ³⁵ S-radiolabelledtranslation products adjusted to 1× HS, 10 mM methionine, mM cysteine(when TRAN³⁵ S-Label was used) and import buffer (1× HS). The sampleswere shaken gently for 30 min at room temperature under fluorescentlights. Import assays can alternatively be carried out using exogenouslyadded ATP instead of light-driven ATP synthesis. Typically, amounts suchas 1 mM-3 mM ATP can be added. Intact chloroplasts were re-isolated forfurther treatment and subfractionation according to the scheme describedby Smeekens et al. (1986) Cell 46:365. After the reaction, thechloroplasts in the import assays were collected by centrifugation at686×g for 3 min, resuspended in 500 μl 1× HS, treated with thermolysin(final concentration 1 μg/μl) and re-isolated through 40% Percollcushions by centrifugation at 1,940×g for 4 min. The intact chloroplastswere collected at the bottom of the tube and resuspended in 500 μl 1×HS, washed once by centrifugation at 686×g for 3 min and resuspended in50 μl of solution A (0.1 M Na₂ CO₃, 0.1 M β-mercaptoethanol). Five μlsamples were removed from each import reaction for chlorophyll analysisas described above. The chlorophyll content was used to normalize thesamples before loading on protein gels. The resuspended chloroplastspellets were then prepared for SDS-PAGE by adding 30 μl solution B (5%(w/v) SDS, 30% (w/v) sucrose, 0.1% (w/v) bromophenol blue) and boiledfor 30 sec. Aliquots from in vitro wheat germ translations and thevarious import reactions were analyzed by SDS-PAGE employing appropriategel density percentages (Laemmli, (1970) Nature 227:80). Afterelectrophoresis, the gels were prepared for fluorography using ENHANCE™(New England Nuclear) according to the manufacturer's instructions andexposed to Kodak XAR™ X-ray film. Import levels and distribution ofimported products were calculated from the fluorograms using an LKBULTRASCAN XL Laser densitometer. The results of the protein fusions aresummarized in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Summary of in vitro import and translation results for various constructs     __________________________________________________________________________    A) Plants                                                                          Translational                                                                       Transit        Protein                                               Promoter Enhancer Signal Passenger Phenotype Level Comments                 __________________________________________________________________________      35SCaMV Rbcs.sup.1 Rbcs Cab.sup.2 + >Cab Enhanced low                               light photosynthesis                                                    35SCaMV Cab Cab Cab 0 =Cab Normal photo-                                            synthesis                                                             __________________________________________________________________________    B) Test tube studies                                                               Translational                                                                       Transit        Import                                                Promoter Enhancer Signal Passenger Translation Level Comments               __________________________________________________________________________      --  Rbcs Rbcs Rbcs high high normal for Rbcs                                  -- Rbcs Rbcs Cab high high 50% higher than                                          Cab alone                                                               -- Cab Cab Cab good/high good normal for Cab                                  -- Cab Cab Rbcs good/high good 50% lower than                                       Rbcs alone                                                              -- Oee1.sup.3 Oee1 Oee1 moderate good normal for Oee1                         -- Rbcs Rbcs Oee1 high high folds greater than                                      Oee1 alone                                                              -- Rbcs Rbcs/ Oee1 high high folds greater than                                 Oee1    Oee1 alone                                                          -- Com44.sup.4 Com44 Com44 low low normal for Com44                           -- Rbcs Com44 Com44 high low normal for Com44                                 -- Rbcs Rbcs/ Com44 high high good import                                       Com44                                                                       -- Rbcs Rbcs Com44 high high good import                                      -- Com70.sup.5 Com70 Com70 low normal normal for Com70                        -- Rbcs Com70 Com70 high normal normal for Com70                              -- PetA.sup.6 PetA PetA low low normal for PetA                               -- Rbcs Rbcs PetA high high high                                              -- Rbcs -- PetA high low none due to loss                                           of signal                                                               -- Rbcs Rbcs/ PetA high high high                                               PetA                                                                        -- Oee1 Oee1 Dhfr.sup.7 moderate good a foreign protein                       -- Dhfr -- Dhfr low no lacks transit signal                                   -- Rbcs Rbcs Dhfr high high high                                              -- Rbcs Rbcs Pka.sup.8 high high higher than Pka                                    itself                                                                  -- Rbcs Rbcs Pkg.sup.9 high high higher than Pkg                                    itself                                                                  -- Pka Pka Pka moderate good normal for Pka                                   -- Pkg Pkg Pkg moderate good normal levels for                                      Pka                                                                     -- Pkg Pkg Rbcs moderate good resembles Pkg                                         levels                                                                __________________________________________________________________________     .sup.1 pea                                                                    .sup.2 pea                                                                    .sup.3 Arabidopsis thaliana                                                   .sup.4 Brassica napus                                                         .sup.5 Spinacea oleracea (spinach)                                            .sup.6 Vicia faba                                                             .sup.7 mouse                                                                  .sup.8 Ricinus cummunis (castor)                                              .sup.9 Nicotiana tabacum (tobacco)                                       

Example 2

Construction of Rbcs-Cab Gene Construct

To produce transgenic tobacco plants with enhanced low lightphotosynthetic capacity through elevation of type I LhcIIb Cab proteinlevels, an enhancement of transcription, mRNA stability, translation andprotein import was attained. The coding portion of the gene constructwas a fusion of a DNA sequence (FIG. 1, SEQ ID NO:1) encoding the matureportion of the type I LhcIIb Cab protein (FIG. 1, SEQ ID NO:2) from pea.The coding sequence for native transit peptide was removed and replacedwith a sequence for the transit peptide from the pea small subunit ofRbc (A. R. Cashmore, in Genetic Engineering of Plants, T. Kosugi, C. P.Meredith, A. Hollaender, Eds. (Plenum Press, New York, 1983) pp. 29-38).The 5' and 3' ends of the type I LhcIIb cab gene sequence used in thepresent construct are shown in FIG. 1. The Rbcs transit peptide (SEQ IDNO:4), and corresponding gene sequence (SEQ ID NO:3), are shown in Table3, together with a short linker sequence linking the transit peptide tothe Cab peptide. A 29 base pair 5' untranslated DNA sequence (5'UTR)originating immediately upstream of the pea Rbcs transit peptide codingregion was used as a translation enhancer. This sequence is shown inTable 3 and is included within SEQ ID NO: 3 (nucleotides 1 to 29).Expression of the gene construct was facilitated by the strong CaMV 35Spromoter (Odell, J. T. et al. (1985) Nature 313:810) and transcriptionaltermination signals originated from the pea Cab gene (A. R. Cashmore(1984) Proc. Natl. Acad. Sci. USA 81:2960. A summary of the geneconstruct is shown in Table 3.

                                      TABLE 3                                     __________________________________________________________________________    Summary of the 35SCAMV-Rbcs-Cab Gene Construct                                __________________________________________________________________________    Key structural parts:                                                           Rbcs (5 untranslated sequence)-pea Rbcs transit peptide-pea Cab protein     body                                                                          Published genetic names of key parts:                                          SS3.6 (5'untranslated sequence)-SS3.6 Rbcs transit peptide-AB80 Cab          protein body                                                                     - Sequence of key parts:                                                     ACGTTGCAATTCATACAGAAGTGAGAAAA ATG GCT TCT ATG ATA TCC                                                        M   A   S   M   I   S                           - TCT TCC GCT GTG ACA ACA GTC AGC CGT GCC TCT AGG GGG CAA TCC GCC            S   S   A   V   T   T   V   S   R   A   S   R   G   N   S   A                 - GCA GTG GCT CCA TTC GGC GGC CTC AAA TCC ATG ACT GGA TTC CCA GTG             A   V   A   P   F   G   G   L   K   S   M   T   G   F   P   V                 - AAG AAG GTC AAC ACT GAC ATT ACT TCC ATT ACA GAC AAT GGT GGA                 K   K   V   Q   T   E   I   T   S   I   T   S   Q   G   G                     - AGA GTA AAG TGC ATG GAT CCT GTA GAG AAG TCT..... (SEQ ID NO:3)              R   V   K   C   M   D   P   L   E   K   S (SEQ ID NO:4)                                 Rbcs  ←                  →  Cab                         - Promoter:                                                                  35S CaMV                                                                       - Terminator:                                                                Cab termination sequences (Cashmore (1984) Proc. Nat. Acad.                   Sci. USA 81:2960-2964)                                                         - Binary vector:                                                             EcoRI-PvuII CAMV-Rbcs-Cab into BamHI/blunt end site of                        pEND4K (kanamycin resistance) Klee et al. (1985)                              Biotechnology 3:637-642).                                                      - Agrobacterium strain:                                                      LBA4404                                                                        - Agrobacterium transformation:                                              Freeze-thaw method (Holsters et al. (1978) Mol. Gen. Genet.                   163:181-187)                                                                   - Transformation protocol:                                                   Leaf disc procedure (Horsh et al. (1985) Science 227:1229-1231)              __________________________________________________________________________

Cloning was initiated by the construction of the pSSTP vector containinga DNA sequence encoding the Rbcs 5'UTR and transit peptide (FIG. 2). TheDNA fragment containing the required components was retrieved fromplasmid pSSNPT (A. R. Cashmore, Univ. Pennsylvania, Philadelphia, Pa.)by digestion with HindIII. Phenol and chloroform:isoamyl alcoholextraction and ethanol precipitation in the presence of 0.1 M NaClfollowed by a 70% ethanol wash were applied after each step of DNAmanipulation as described in Example 1 to inactivate enzymes and toconcentrate the DNA. The DNA precipitate was collected bycentrifugation, dried and redissolved in 10 μl water. The HindIII endwas rendered blunt utilizing the Klenow fragment of E. coli DNApolymerase I (Promega). The reaction consisted of 1 unit of Klenow, 0.1mM each of dATP, dCTP, dGTP and dTTP, 50 mM Tris-HCl pH7.5, 10 mM MgCl₂,5 mM DTT, and the DNA from the above step, and was incubated at 37° C.for 1 h. After repurification by organic solvent extractions, the DNAwas digested with BamHI, separating the required DNA fragment from therest of the PSSNPT plasmid. The HindIII-BamHI DNA fragment was gelpurified and ligated into the SmaI and BamHI sites of pGEM4 (Promega)that had been cleaved and subsequently dephosphorylated by calfintestinal alkaline phosphatase (Pharmacia). The purification of DNA wascarried out using the standard low melting agarose gel and phenolextraction method (Sambrook et al. 1989, supra.). The low meltingagarose was purchased from BRL (Gaithersburg, Md., USA). DNA wasrecovered from appropriate low melting agarose slices by heating at 65°C. followed by extraction with phenol, prewarmed initially at 37° C.,and centrifugation. The phenol extraction was repeated and the aqueousDNA layer was then adjusted to 0.1 M NaCl and centrifuged for 10 min ina microfuge. The supernatant was subjected to chloroform:isoamyl alcoholextraction followed by precipitation in ethanol as described above. TheDNA pellet was collected by centrifugation, washed with 70% ethanol,dried and resuspended in water.

The mature type I LhcIIb Cab coding DNA sequence (pea AB80), containedin a XbaI-PstI DNA fragment, was retrieved by digesting plasmid pDX80(A. R. Cashmore, Univ. Pennsylvania, Philadelphia, Pa.) with XbaI andPstI (FIG. 3). The DNA fragment was also gel purified and inserted intothe plasmid vector pSSTP (FIG. 2) via the XbaI and PstI sites. Prior toligation, these sites had been dephosphorylated by adjusting therestriction digestion reaction with 3.5 μl 1M Tris-HCl, pH 8.0 andadding 0.5 units of calf intestinal alkaline phosphatase. Following aminute incubation at 37° C., the dephosphorylated vector was repurifiedby organic solvent extraction and precipitated with ethanol. Theresulting plasmid was designated PRBCS-CAB (FIG. 3).

The Rbcs-Cab chimeric gene was fused to the 35S CaMV constitutivepromoter by inserting a gel-purified EcoRI-HindIII fragment carrying the35S CaMV promoter from plasmid pCAMV (A. R. Cashmore, Univ.Pennsylvania, Philadelphia, Pa.) into the EcoRI-Asp718 sites ofpRBCS-CAB (FIG. 4). The corresponding HindIII and Asp718 restrictionsites were made blunt using the Klenow fragment of DNA polymerase I. The35S CaMV-Rbcs-Cab construct was then transferred as an EcoRI-PvuII DNAfragment to the BamHI site of the binary vector pEND4K (FIGS. 5 and 6)(Klee, H. et al. (1985) Biotechnology 3:637). All of the restrictionenzyme-generated ends were made blunt by Klenow fragment in this step.

All ligation steps were carried out at 15° C. overnight using T4 DNAligase. The ligation reactions consisted of the two appropriate targetDNA molecules, ligase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mMDTT, 1 mM ATP) and 1-3 units of enzyme. All appropriate steps of thegene construction process were introduced into bacteria using standardCaCl₂ bacterial transformation protocol (Sambrook et al. 1989, supra)and the E. coli host strain HB101. All recombinant plasmids werepropagated in HB101 and isolated using standard techniques (Sambrook etal. 1989, supra). The resolution of DNA fragments was facilitated byusing standard agarose and polyacrylamide gel electrophoresistechniques.

Example 3

Transformation and Selection of Plants

The pEND4K-CAMV-Rbcs-Cab plasmid was introduced into Agrobacterium usingthe freeze-thaw method (Holsters et al. (1978) Mol. Gen. Genet.163:181-187). Competent LBA4404 cells were obtained by inoculating 50 mlof LB broth (50 μg/ml rifampicin) with 500 μL of an overnight culture,followed by incubation at 28° C. with vigorous shaking until the celldensity measured ⁰.7_(OD) at 650 nm. Cells were harvested bycentrifugation at 2000×g for 5 min at 4° C., washed in ice cold 0.1MCaCl₂ and resuspended in 1 ml of ice cold 20 mM CaCl₂. A 150 μl aliquotof competent LBA4404 cells was mixed with 1 μg of plasmid DNA in amicrofuge tube, and immediately frozen in liquid nitrogen. These cellswere incubated at 37° C. in a water bath or thermostat block for 5 min,1 ml of LB broth added, and the mixture incubated at 28° C. with shakingfor 3 h. The cells were recovered by centrifugation at 2000×g for 5 minand resuspended in 100 μl of LB broth. Cells were plated on LB platescontaining 100 μg/ml kanamycin and 50 μg/ml rifampicin and incubated for2 days at 28° C. The presence of the pEND4K-CAMV-Rbcs-Cab plasmid wasconfirmed by Southern blot analysis of plasmid preparations obtainedfrom single kanamycin-resistant colonies. Three ml of YEB brothcontaining 50 μg/ml rifampicin and 100 μg/ml kanamycin were inoculatedwith a kanamycin-resistant colony and incubated overnight at 28° C. withshaking. The overnight culture (1.5 ml) was centrifuged for 30 sec in amicrofuge. The cells were resuspended in 0.1 ml of GTE solution (50 mMglucose, 10 mM Na₂ EDTA, 25 mM Tris-HCl pH 8.0) with 4 mg/ml oflysozyme, and incubated at room temperature for 10 min. Phenol (30 μl)previously equilibrated with 2 vols of 1% (w/v) SDS, 0.2N NaOH wasadded. The mixture was vortexed gently until viscous and incubated atroom temperature for 10 min. The lysed cells were neutralized with 3Msodium acetate, pH 4.8 (150 μl) and incubated at -20° C. for 15 min.before the mixture was centrifuged for 3 min in a microfuge. Thesupernatant was transferred to a fresh microfuge tube, two volumes ofethanol added, and the mixture was incubated at -80° C. for 15 min toprecipitate the DNA. Following centrifugation, the DNA pellet wasresuspended in 90 μl of water. Ten μl of 3M sodium acetate pH 7.0 wereadded, followed by an equal volume of phenol/chloroform, and the mixturewas vortexed. After centrifuging for 5 min in a microfuge, thesupernatant was transferred to a fresh tube and the DNA precipitated byadding 2 volumes of 100% ethanol. After centrifugation, the pellet waswashed with 70% ethanol, dried and resuspended in 50 μL of TE (10 mMTris-HCl pH 8.0, 1 mM Na₂ EDTA).

The integrity of the pEND4K-CAMV-Rbcs-Cab plasmid in Agrobacterium wasverified by restriction and Southern blot analysis of the plasmidisolated as described above and in Sambrook et al. 1989, supra. One ofthe Agrobacterium selected colonies containing an intactpEND4K-CAMV-Rbcs-Cab was used for plant transformation.

Tobacco plants were transformed with plasmid pEND4K-CAMV-Rbcs-Cabfollowing the leaf disc transformation protocol essentially as describedby Horsch et al. (1985) Science 227:1229). Only young, not fullyexpanded leaves, 3-7" length, from one month old plants were used.Excised leaves were surface-sterilized in 10% (v/v) sodium hypochlorite,0.1% (v/v) Tween and rinsed 4 times with sterile deionized water. Fromthis point on, standard aseptic techniques for the manipulation of thesterile material and media were used. Leaf discs, 6 mm in diameter, weremade with the aid of a sterile paper punch and incubated for 10-20 minin a 1:5 dilution of an overnight culture of Agrobacterium harbouringthe pEND4K-CaMV-Rbcs-Cab construct. After inoculation, excess bacteriawere removed from the discs by briefly blotting on sterile filter paperand the discs transferred to petri dishes containing "shoot medium"(Horsch et al. (1988) in Plant Molecular Biology Manual, (Eds. S. B.Gelvin, R. A. Schilperoort) Kluwer Acad. Publishers, A5:1-9). Petriplates were sealed with parafilm and incubated in a growth chamber (24°C. and equipped with "grow" mixed fluorescent tubes). After two days,Agrobacterium growing on the discs were killed by washing in 500 mg/mlCefotaxime in liquid "shoot medium" and the discs were transferred tofresh "shoot medium" containing 500 mg/ml Cefotaxime and 100 mg/mlkanamycin to select for growth of transformed tobacco cells.

Leaf discs were incubated under the same growth conditions describedabove for 3-5 weeks, and transferred to fresh medium on a weekly basis.During this period of time, approximately 40 green shoots emerging fromthe 60 discs were excised and transferred to "root medium" (Horsch etal. (1988) supra) containing 100 μg/ml kanamycin. Shoots which rooted inthe presence of kanamycin and were verified to possess high levels ofNptII activity (McDonnell, R. E. et al. (1987) Plant Mol. Biol. Rep.5:380) were transferred to soil. Selected transformants were selfed andseeds collected. T1 seeds from seven transgenic tobacco lines displayinghigh levels of NptII activity were propagated at low light parameters(50-100 μmol·m⁻² ·s⁻¹) to determine which lines contained high levels ofsteady state transgene mRNA.

The same construct has been introduced into two cultivars ofArabidopsis, three cultivars of Brassica, tomato, lettuce and alfalfa.All of these species demonstrate increased growth in culture compared totheir wild-type counterparts, especially under low light intensities.These plants have a better shade avoidance response. They grow faster,bigger and seek light more responsively than their wild-typecounterparts. This is evident at 65 μmoles/meter² /sec of illuminationin tobacco and lettuce, and 5 μmole/meter² /sec of illumination forArabidopsis.

Example 4

RNA Analysis

Isolation of total RNA and subsequent blot hybridization analyses werecarried out as described in A. R. Cashmore, 1982 in Methods inChloroplast Molecular Biology, M. Edelman, R. B. Hallick, N. H. Chua,Eds. (Elsevier Biomedical Press, pp. 533-542) and Maniatis, T., Fritsch,E. F., and Sambrook, J., Molecular Cloning: A Laboratory Manual, (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982)). Total RNAwas isolated from twenty individual T1 plants from each primarytransformant. Leaf samples were collected between 11:00 a.m. and 1:00p.m. Formaldehyde denaturing gels were used to resolve RNA andtransferred onto nitrocellulose as described (Sambrook et al. 1989,supra). The RNA blot (FIG. 7A) was probed with the pea Cab DNA probe(marked PROBE 1), stripped and rehybridized with the pea Rbcs transitpeptide-specific DNA probe (marked PROBE 2). Numbers 1-7 indicate theprimary transformants carrying the Rbcs-Cab transgene and exhibitinghigh levels of NptII activity. Plants 7-12 represent plants that havebeen transformed with a control pea Cab construct. Plant 13 represents awild-type nontransformed tobacco plant (Nicotiana tabacum cv. PetitHavana SR1). Transcript levels detected by the pea Cab DNA probe werenormalized and quantitated by laser densitometry. Plants arising fromtransformant lines 3 and contained the lowest amount of pea cab mRNA,whereas lines 1, 2 and 7 contained the highest levels. The same resultswere obtained with the pea Rbcs transit peptide-specific DNA probe (FIG.7A). Total RNA from wild-type plants (WT) did not hybridize to either ofthe DNA probes.

Individual T1 plants from the lines with the highest observed transgenemRNA levels were self-crossed and subjected to segregation analysis. Thetransgene mRNA levels of subsequent homozygous lines were analyzed inthe same manner as the primary transformant lines (FIG. 7B). Numbersindicate the derivation of the homozygous lines. Seeds were germinatedat 24° C. on moist filter paper, transferred to pots containing amixture of soil and vermiculite (1:1) and propagated in growth chambersset at 50-100 μmol·m⁻² ·s⁻¹ lighting with a 14 h light/10 h darkphotoperiod and 24/18° C. day/night temperature. The pots were watereddaily with a complete nutrient solution containing 10 mM nitrate and 2mM ammonium. Several subsequent homozygous lines exhibited high levelsof transgene mRNA and displayed the same phenotype.

Example 5

Protein and Chlorophyll Analysis

Thylakoid protein profiles of homozygous lines derived from transgenicline 2 (TR) were compared to wild-type (WT) to determine if additionaltransgene transcripts translated into an overall increase in steadystate Cab levels. Nine different leaf disc samples were collected fromhomozygous transgenic lines derived from primary transformant 2 (markedTR) and from wild-type plants (marked WT). Samples were collectedbetween 11:00 a.m. and 1:00 p.m. Thylakoids were isolated (K. E.Steinback, et al., in Methods in Chloroplast Molecular Biology, M.Edelman, R. B. Hallick, N. H. Chua, Eds. (Elsevier Biomedical Press,Amsterdam, 1982) pp. 863-872; Vernon, L. P. (1960) Anal. Chem. 32:1144)and analyzed using standard immunoblotting techniques. Cab/oeel ratioswere determined by laser densitometry of the corresponding immunostainedbands. The bands corresponding to Oeel and Cab are marked. Increases inoverall Cab protein levels were detected by simultaneously probing theblots with antibodies against the 33 kDa protein of the PSIIoxygen-evolving complex (Oee1) and Cab (FIG. 7C). The ratio of Cab toOee1 was used to determine Cab levels relative to PSII units by usingOee1 as an internal marker of PSII levels. The densitometry resultsindicated that the level of Cab protein is enhanced 2-3× relative toOee1, suggesting that there is more Cab protein per PSII. Parallelenhancement of chlorophyll content was evident when the LhcII complexeswere isolated (K. E. Steinback, (1982) supra; L. P. Vernon (1960)supra). Approximately 1.5× more chlorophyll and about 2-3× more LhcIIcomplexes were recovered per gram of fresh weight leaf, indicating thatthe additional Cab proteins are functionally binding chlorophyll.

Example 6

Morphological and Developmental Analysis

The TR plants, both primary transformants and subsequent selfedhomozygous lines, exhibit growth and morphological differences relativeto WT under all conditions tested, e.g., greenhouse or growth chambers.All plants shown were at the same developmental age and were propagatedas described above. The TR plants display a higher level of vigor underlow light regimes (50-80 μmol·m⁻² ·s⁻¹) (FIG. 8A).

The high light responses of the TR plants are enhanced. They producemore biomass and more robust growth patterns, depending on the intensityof the lighting conditions during propagation. The TR plants are biggerthan their WT counterparts under high light intensities, such as ingreenhouses. Among other characteristics, TR plants, compared to WTplants show increased stem girth and less variability in growth pattern.Further, field trials show that TR plants grow as well as WT plants infull sunlight field conditions in terms of biomass and size. Nodetrimental effects were observed in TR plants under these conditions.

The elevation of Cab appears to induce a series of changes, the mostprominent ones being broader leaves with a smooth blade, a continuousedge around the leaves, higher vegetative biomass and delayed floweringtime. In addition to the overall enlargement of leaf size, the base ofthe petiole is more expanded relative to the WT leaves (the 7th fullydeveloped leaf from both WT and TR were compared) (FIG. 8B). The TRleaves are thicker with relatively larger intercellular spaces (FIG.8C). The light micrographs (FIG. 8C) represent samples from theintermediary area of the leaf blade. Leaf pieces were fixed in FAA50 andexamined using a light microscope (D. A. Johansen, Plant Microtechnique,(McGraw-Hill Book Co., New York, 1940)).

Leaf samples were selected as above and processed for electronmicroscopy by fixing in 2.5% glutaraldehyde buffered with 0.1M phosphatebuffer (pH 7.5) and post-fixing with 1% osmium tetroxide for 2 h.Following dehydration in an ethanol series, the leaf samples wereembedded in Spurr resin, sectioned and further stained in uranyl acetateand plumb citrate (Spurr, A. R. (1969) J. Ultrastr. Res. 26:31;Reynolds, E. S. (1963) J. Cell Biol. 17:208; Watson, M. L. (1958) J.Biophys. Biochem. Cytol. 4:475). The same scale in the applies to bothWT and TR mesophyll tissue photographs. The TR leaf cells contain highernumbers of chloroplasts on a per cell basis and the plastids are largerwith a strikingly rounder shape (FIGS. 11A-D). Differences in theinternal organization of the plastids, e.g. stacking of the thylakoids,were not detected at the level of resolution used. Differences were notdetected at any level with respect to the mitochondria, vacuole ornucleus.

The germination rate of TR seeds was strikingly different than that ofWT seeds. TR seeds germinate on average 1-3 days earlier than WT seedson solid MS media (FIG. 9), and the newly emerged TR seedlings arealready green and grow faster upon emergence than WT. The WT seedlingsemerge yellowish and begin greening within the day. Callus tissuecomprising TR cells grows 2-3× faster than callus comprising WT cells(FIGS. 10A-B).

Transplants of TR plants also withstand transplant shock better thantransplants of WT plants. They recover and establish normal growingpatterns more rapidly.

Example 7

Physiological and Biochemical Analysis

Functionality and enhancement of photosynthetic activity as a result ofthe extra Cab protein was assessed using four different criteria:

1) Gas exchange characteristics;

2) Metabolite level changes;

3) Carbohydrate content; and

4) PSII electron transport efficiency.

Photosynthetic rates of TR and WT plants propagated under limiting lightconditions were compared. Plants were cultivated under two differentlight intensities, 50 μmol·m⁻² ·s⁻¹ (referred to as low, (FIG. 12A) and500 μmol·m⁻² ·s⁻¹ (referred to as high, (FIG. 12B). Photosynthesis wasmeasured using a leaf disc oxygen electrode (LD2/2 Hansatech, UK) undersaturating 5% CO₂ at 25° C. The 5% CO₂ was supplied from 200 μl of a 2MKHCO₃ /K₂ CO₃ mixture (pH 9.3) on felt in the base of the leaf discelectrode (Walker, D. A. (1987) The use of the oxygen electrode andfluorescence probes in simple measurements of photosynthesis Universityof Sheffield, Sheffield, U. K.). Illumination was provided by a slideprojector Novomat 515 AF (Braun, Germany). The data are means of 5plants of each phenotype. Standard deviations were less than 10% of themeans.

Photosynthetic response curves of TR plants display a behavior distinctfrom WT plants (FIG. 12A). In low light (between 20-100 μmol·m⁻² ·s⁻¹),the TR plants exhibit a higher rate of photosynthesis than in WT plants;whereas, the reverse situation occurs in higher light intensities (FIG.12A). As the light intensity increases, the response curves become moresimilar, intersecting at approximately 300 μmol·m⁻² ·s⁻¹, where TRtissue reaches saturation at a lower rate. At the same light intensity,the increase in photosynthesis is higher and has not reached saturationin WT tissue. Saturation in WT tissue occurs at about 450 μmol·m⁻² ·s⁻¹.

The same response was displayed by plants grown in higher irradiance(500 μmol·m⁻² ·s⁻¹) (FIG. 12B). The rate in TR tissue is higher than WTtissue in the range of 20-500 μmol·m⁻² ·s⁻¹, reaching saturation inhigher light intensities, while WT remains unsaturated at 1000 μmol·m⁻²·s⁻¹. The increased low light photosynthetic capacity of TR tissue wasalso evident in air CO₂ levels and at a light intensity of 100 μmol·m⁻²·s⁻¹, where TR tissue exhibited an average photosynthetic rate 50%higher than WT tissue (3.3±0.8 vs. 2.2±0.8 μmol O₂ ·m⁻² ·s¹,respectively).

Alterations in metabolite and adenylate levels are also indicators ofchanges in photosynthetic capacity. In low light, photosynthesis ismainly limited by the capacity of electron transport to generate ATP andNADPH, the assimilatory force FA (Heber, U. et al. (1986) Biochim.Biophys. Acta 852:144; Heber et al., in Progress in PhotosynthesisResearch, J. Biggins, Ed., (Martinus Nijhoff, Dordrecht) Vol. 3 (1987)pp. 293-299). The strength of F_(A) can be estimated by the ratio of PGA(3-phosphoglyceric acid) to TP (triose phosphate) (Dietz, K. J. andHeber, U. (1984) Biochim. Biophys. Acta. 767:432); ibid 848:392 (1986)).Measurements were obtained under 100 and 1000 μmol·m⁻² ·s⁻¹ lighting,and in 850 μbar external CO₂ concentration to minimize the effects ofphotorespiration (Table 4). When photosynthesis achieved steady state,the leaves were freeze-clamped and prepared for metabolite extraction.The CO₂ assimilation rate of TR leaves in 100 μmol·m⁻² ·s⁻¹ lighting was53% higher than WT leaves. The levels of PGA were similar between TR andWT plants, however, the TP level was 33% higher in TR. Thus, the PGA/TPratio is higher in WT plants, indicating a limitation in the reductionof PGA to TP by the supply of ATP and NADPH in WT plants. The changes inadenylates indicate that the ATP content in TR leaves was twice thevalue observed in WT leaves, whereas the ADP content in both plants wassimilar. The ATP/ADP ratio in the chloroplast is lower than in thecytosol, typically calculated to be between 1.5 and 3.0 (Stitt, M. etal. (1982) Plant Physiol. 70:971; Giersch, C. et al. (1980) Biochim.Biophys. Acta 590:59; Neuhaus, N. E. and Stitt, M. (1989) Planta179:51). As light intensity increases, the ratio decreases even further(Dietz and Heber, supra ). The ATP/ADP ratio is higher in TR plants thanWT plants (2.2 vs. 0.8). These results indicate that TR plants have anincreased capacity to generate ATP in low light, leading to anenhancement of PGA reduction and a higher photosynthetic rate.

Changes in the level of hexose phosphate were also observed, with morehexose phosphate in WT than TR plants. The G6P/F6P ratio is an indicatorof hexose distribution in a cell, with values of 1-2 indicatingchloroplastic compartmentalization and predominantly starch synthesis,and ratios of 3-5 indicating a cytoplasmic location with sucrosesynthesis being dominant (Gerhardt, R. et al. (1987) Plant Physiol.83:399). Thus, the low G6P/F6P values for both WT and TR plants grown inlow light indicate that the carbon fixed is being partitioned mainlyinto starch.

The enhanced capacity to absorb light has a negative effect onphotosynthetic metabolism of TR plants in high irradiance regimes. Thephotosynthetic rate of WT plants was instead 37% higher than that of TRplants. The changes in metabolite levels and ratios indicate that majoralterations in the regulatory mechanisms of photosynthesis have occurredin TR plants to compensate for the enhanced light-absorbing capacity inhigh light. The PGA/TP ratio was identical in both plants and theATP/ADP ratio was lower in TR plants indicating thatphotophosphorylation was limiting photosynthesis. The change inpartitioning indicated by the G6P/F6P ratio increase (2.9 vs. 3.9 in WTand TR plants, respectively) points to an increase in sucrose synthesisto compensate for the elevated demand for inorganic phosphate (Pi) in TRplants. The TR plants appear to be less efficient in the recycling of Pivia sucrose synthesis in high light.

                                      TABLE 4                                     __________________________________________________________________________    Photosynthesis and metabolite content in plants grown under 50 μmol        · m.sup.-2 · s.sup.-1 lighting                                            100 μmol · m.sup.-2 · s.sup.-1                                                              CER Metabolite Content                                                       (nmol · mg.sup.-1                                                     Chl.) Metabolite Ratio                                                       (mol/mol)                     Plant Type                                                                          (μmol · m.sup.-2 · s.sup.-1)                                     PGA  TP  ATP  ADP  G6P  F6P  G1P  PGA/TP                                                                             ATP/ADP                                                                            G6P/F6P             __________________________________________________________________________      Wild-type 1.7 ± 0.1 364 ± 110 46 ± 21  74 ± 27 89 ± 51                                                                 92 ± 39 98                                                                 ± 40 69 ±                                                               48 8.4 0.8 0.9                                                                 Transgenic 2.6                                                               ± 0.6 353                                                                  ± 47  61                                                                   ± 8  181                                                                   ± 38 85 ±                                                               32 43 ± 9 65                                                               ± 38 61 ±                                                               9  5.8 2.2 0.7      1000 μmol · m.sup.-2 · s.sup.-1                          Wild-type                                                                           8.8 ± 0.7                                                                          320 ± 174                                                                       92 ± 49                                                                        112 ± 43                                                                        86 ± 37                                                                         174 ± 62                                                                        59 ± 7                                                                          40 ± 9                                                                          3.5  1.2  2.9                   Transgenic 6.4 ± 1.8 259 ± 54  73 ± 17  89 ± 32 150 ± 49                                                                217 ± 66 56                                                               ± 27 40 ±                                                               23 3.5 0.6          __________________________________________________________________________                                                              3.9                  External CO.sub.2 concentration was 850 μl · L - 1 and leaf       temperature was 25° C.                                                 The data presented are averages of 4 plants ± SD.                          The rates of CO.sub.2 assimilation were measured in an open gas exchange      system using a Binos100 (Rosemount, Germany) infrared gas analyser.           The leaf chamber was designed and built with aluminum alloy to allow rapi     acquisition of leaf disc samples and quick freezing.                          The lower side of the chamber was sealed with Parafilm, through which a       pneumatically driven liquid N.sub.2frozen copper rod could enter.             The upper side of the chamber contained a clear acrylic ring to stop the      leaf disc cutter.                                                             The frozen leaf discs (8 cm.sup.2) were stored in liquid N.sub.2 until        use.                                                                          Actinic illumination was provided from two branches of the fiber optic        from a KL1500 cold light source (Schott, Maiz Germany) directed onto the      top of the chamber at a 45° C. angle.                                  Chamber temperature was controlled by circulating water through the           chamber cavity.                                                               The water vapor deficit of the incoming air was readjusted to 18 mbar by      passing the air stream to a coil immersed in a water bath kept at             15° C.                                                                 Leaf samples were extracted in 10% (v/v) HClO.sub.4 and the indicated         metabolites were determined using a Hitachi U3300 (Tokyo, Japan)              spectrophotometer (Labate, C. A. and R. C. Leegood, R. C. (1989) Plant        Physiol. 91:905; Lowry, O. H. and Pasonneau, J. V. (1972) A flexible          system of enzymatic analysis (Academic Press, New York).                 

Changes in photosynthetic activity were also reflected in carbohydratecontent. Starch and sucrose contents were relatively balanced with nomajor partitioning changes in young leaves of TR or in WT plants underboth lighting regimes (50 and 500 μmol·m⁻² ·s⁻¹) (Table 5A). Sucrose andstarch were produced in equal amounts except that the total carbohydratecontent in TR plants was 2-3× higher than in WT plants. The seeds of TRplants contained 2× more starch than WT seeds when grown under low lightand then shifted to high light at later stages of growth. The totalcarbohydrate level of TR and WT plants appeared unaffected by lightingchanges in the young leaves.

A similar situation occurs in fully developed WT leaves (Table 5B), withstarch and sucrose levels remaining fairly balanced and largelyindependent of lighting. The overall carbohydrate levels, however, werehigher than in young leaves. TR leaves at the same developmental stagereact differently to the two lighting regimes by varying the starch andsucrose levels. Sucrose levels were higher in low light, whereas starchwas greater in high irradiance. Although the total carbohydrate levelsincrease substantially in high light in both TR and WT leaves, the levelin TR leaves was approximately 49% higher than in WT leaves.

                  TABLE 5                                                         ______________________________________                                        Carbohydrate content in young and fully developed leaves                      ______________________________________                                        (A) Starch and sucrose content in young leaves.                                 The data are averages of 4 plants ± SD.                                            Starch     Sucrose  Total Carbohydrate                              Plant Type                                                                              μmol hexoses equivalents · mg - 1Ch1                    ______________________________________                                        Plants cultivated under 50 μmol · m.sup.-2 s.sup.-1                   Wild-type 0.6 ± 0.2                                                                             0.8 ± 0.1                                                                         1.4 ± 0.3                                    Transgenic 1.3 ± 0.2 1.6 ± 0.4 3.0 ± 0.6                           Plants cultivated under 500 μmol · m.sup.-2 s.sup.-1                  Wild-type 0.6 ± 0.3                                                                             0.4 ± 0.1                                                                         1.0 ± 0.4                                    Transgenic 1.8 ± 0.5 1.4 ± 0.4 3.2 ± 0.9                           ______________________________________                                        (B) Starch and sucrose content in fully developed leaves.                       The data are averages of 6 plants ± SD.                                            Starch     Sucrose  Total Carbohydrate                              Plant Type                                                                              μmol hexoses equivalents · mg - 1Ch1                    ______________________________________                                        Plants cultivated under 50 μmol · m.sup.-2 s.sup.-1                   Wild-type 1.2 ± 0.4                                                                             1.8 ± 1.2                                                                         3.0 ± 1.6                                    Transgenic 1.2 ± 0.5 2.1 ± 1.4 3.3 ± 1.9                           Plants cultivated under 500 μmol · m.sup.-2 s.sup.-1                  Wild-type 4.1 ± 1.9                                                                             3.8 ± 1.9                                                                         7.9 ± 3.8                                    Transgenic 7.0 ± 2.8 4.8 ± 1.8 11.8 ± 4.6                          ______________________________________                                         Soluble sugars were assayed spectrophotometrically following the              extraction of leaves in HClO.sub.4.                                           For estimation of starch, the insoluble leaf extracts were washed with        0.5M MESHCl (pH 4.5), resuspended in 0.5 ml of the same buffer, and           digested with an amylase (4 units ml - 1)amyloglucosidase (14 units ml -      1) cocktail.                                                                  After centrifugation, the supernatants were assayed for glucose (Jones, M     G. K. et al. (1977) Plant Physiol. 60:379).                              

FIG. 13 shows the light response curves for qP (FIG. 13A), qN (FIG.13B), Fv/Fm (FIG. 13C) and .O slashed._(PSII) (FIG. 13D) measured in airfor WT and TR plants grown for 6-8 weeks postgermination. The data aremeans of 4 plants of each phenotype. Standard deviations were less than5% of the means. Chlorophyll fluorescence was analyzed using a pulseamplitude modulation fluorometer (PAM101, Heinz Walz, Effeltrich,Germany). Fo (base fluorescence in dark) was measured in leavesdark-adapted (for a period of 30-60 min) using a weak modulatedmeasuring light (approximately 1 μmol·m⁻² ·s⁻¹) provided by a fibreoptic probe located under the lower window of the leaf chamber(approximately 5 mm from the leaf surface), which also collected thefluorescence signal. For the determination of maximal fluorescence (Fm),a saturating pulse of light (7500 μmol·m⁻² ·s⁻¹), activated by a PAM103trigger control unit, was applied at a frequency of 30 s and a durationof 1 s. The steady state fluorescence yield (Fs) was monitored followingthe onset of illumination with actinic light. The photochemical (qP) andnon-photochemical (qN) quenching parameters were determined according toSchreiber et al. (1986) Photosynth. Res. 10:51. The quantum efficiencyof electron flux through PSII .O slashed._(PSII) was determined by theproduct of qP and the efficiency of excitation capture by open PSIIreaction centers (Fv/Fm) (Genty et al. (1989) Biochim. Biophys. Acta.990:87).

The effect of extra Cab protein on electron transport efficiency by PSII(.O slashed._(PSII)) in response to varying irradiance was determined bymeasuring chlorophyll fluorescence characteristics (Genty, et al. (1989)supra) in air at steady state photosynthesis (FIGS. 13A-D). Measurementswere carried out with plants propagated in low irradiance (50 μmol·m⁻²·s⁻¹). There was a corresponding decrease in .O slashed._(PSII) as lightintensity increased (FIG. 13D), with a less pronounced decline in TRplants. The quantum efficiency of open PSII reaction centers (Fv/Fm)also decreased with increasing light intensity; however, TR plantsdisplayed a different pattern from 100 to 600 μmol·m⁻² ·s⁻¹ (FIG. 13C).The Fv/Fm decline in TR plants was less substantial than in WT plantsbetween this range of light intensities, becoming more similar to WTplants above this range. The efficiency of .O slashed._(PSII) can bedetermined by the product of Fv/Fm and the photochemical quenching ofchlorophyll fluorescence (qP). The qP value reflects the oxidized stateof the primary acceptor Q_(A) (Schreiber, U. et al. (1986) supra). Adecrease in qP as light intensity increased was observed (FIG. 13A). TheTR plants remain significantly more oxidized than WT except at highirradiance. The chlorophyll fluorescence data also indicate thatexposure of TR plants to high irradiance does not lead tophotoinhibition since Fv/Fm ratios were similar for TR and WT plants.

Non-photochemical quenching (qN) was higher in WT from 100 to 600μmol·m⁻² ·s⁻¹ (FIG. 13B). It is evident that in TR the regulation ofPSII function was affected by the enhanced light-absorbing capacity. Thehigher efficiency flow of low light electron transport by PSII in TRappears to be attributable mainly to a higher Fv/Fm and a lower qN.Conversely, photochemical quenching (qP) becomes the main factordetermining the higher efficiency of PSII (.O slashed._(PSII)) in TRunder light intensities higher than 600 μmol·m⁻² ·s⁻¹.

These data show that elevating type I LhcIIb Cab protein levels bygenetic manipulation results in measurable and significant changes to aplant. LhcII is believed to play a key role in controlling theproportion of absorbed excitation energy directed to PSII. Normally,photosynthesis would be limited in low light by the capacity of electrontransport to generate ATP and NADPH for the reduction of CO₂ ; however,the enhanced level of Cab protein allows the TR plants to channel moreenergy through the electron transport system under light limitingconditions. The presence of elevated LhcIIb Cab proteins could alsocontribute to changes in reductant and excitation pressures ofphotosynthesis.

The higher photosynthetic capacity of TR plants is independent of thecultivation lighting parameters. Transgenic plants grown either in lowor intermediary irradiance display similar low light photosyntheticcapacities, as evident in the higher initial slope of the light responsecurves exhibited in both saturating and air levels of CO₂ (FIGS. 12A-12Dand 13A-13D). This suggests higher ATP and NADPH generation capacity asreflected by the metabolite ratio changes at steady statephotosynthesis. Lower PGA/TP and higher ATP/ADP ratios in TR plantsindicate that elevating Cab protein resulted in increased ATP synthesis,thereby enhancing PGA reduction. The chlorophyll fluorescence parametersalso suggest more efficient electron transport in TR plants. Theefficiency of excitation energy capture by open PSII centers (the Fv/Fmratio) was higher in TR plants below 500 μmol·m⁻² ·s⁻¹ illumination,with a related decrease in non-photochemical quenching (qN) compared toWT plants under the same conditions. The enhanced light-gatheringcapacity is associated with anatomical modifications to the leaves,e.g., enlargement of intercellular spaces, which probably contribute toincreased CO₂ diffusion towards the chloroplasts, facilitating higherrates of photosynthetic and carbohydrate synthesis.

All citations in this application to materials and methods are herebyincorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1166 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION:184..990                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - TCCATGAACG GATTCTAGAA TTGCAAAGAA AATCTCCAAC TAGCCATAGC TT -            #TAGATAAC     60                                                                 - - ACACGATAAG AGCATCTGCA TTATAAATAC AGACTCATAT TCATCTTACA AA -            #ATCACCAT    120                                                                 - - TGATAAGGAT ACAATTATCA AAAGCATAAC AATCTTTTCA ATTTCATTGC AA -            #TATAATAC    180                                                                 - - ACG ATG GCC GCA TCA TCA TCA TCA TCC ATG GC - #T CTC TCT TCT CCA        ACC      228                                                                        Met Ala Ala Ser Ser Ser Ser Ser - #Met Ala Leu Ser Ser Pro Thr                 1            - #   5               - #   10               - #   15       - - TTG GCT GGC AAG CAA CTC AAG CTG AAC CCA TC - #A AGC CAA GAA TTG GGA          276                                                                       Leu Ala Gly Lys Gln Leu Lys Leu Asn Pro Se - #r Ser Gln Glu Leu Gly                            20 - #                 25 - #                 30              - - GCT GCA AGG TTC ACC ATG AGG AAG TCT GCT AC - #C ACC AAG AAA GTA GCT          324                                                                       Ala Ala Arg Phe Thr Met Arg Lys Ser Ala Th - #r Thr Lys Lys Val Ala                        35     - #             40     - #             45                  - - TCC TCT GGA AGC CCA TGG TAC GGA CCA GAC CG - #T GTT AAG TAC TTA GGC          372                                                                       Ser Ser Gly Ser Pro Trp Tyr Gly Pro Asp Ar - #g Val Lys Tyr Leu Gly                    50         - #         55         - #         60                      - - CCA TTC TCC GGT GAG TCT CCA TCC TAC TTG AC - #T GGA GAG TTC CCC GGT          420                                                                       Pro Phe Ser Gly Glu Ser Pro Ser Tyr Leu Th - #r Gly Glu Phe Pro Gly                65             - #     70             - #     75                          - - GAC TAC GGT TGG GAC ACT GCC GGA CTC TCT GC - #T GAC CCA CAG ACA TTC          468                                                                       Asp Tyr Gly Trp Asp Thr Ala Gly Leu Ser Al - #a Asp Pro Gln Thr Phe            80                 - # 85                 - # 90                 - # 95       - - TCC AAG AAC CGT GAG CTT GAA GTC ATC CAC TC - #C AGA TGG GCT ATG TTG          516                                                                       Ser Lys Asn Arg Glu Leu Glu Val Ile His Se - #r Arg Trp Ala Met Leu                           100  - #               105  - #               110              - - GGT GCT TTG GGA TGT GTC TTC CCA GAG CTT TT - #G TCT CGC AAC GGT GTT          564                                                                       Gly Ala Leu Gly Cys Val Phe Pro Glu Leu Le - #u Ser Arg Asn Gly Val                       115      - #           120      - #           125                  - - AAA TTC GGC GAA GCT GTG TGG TTC AAG GCA GG - #A TCT CAA ATC TTT AGT          612                                                                       Lys Phe Gly Glu Ala Val Trp Phe Lys Ala Gl - #y Ser Gln Ile Phe Ser                   130          - #       135          - #       140                      - - GAG GGT GGA CTT GAT TAC TTG GGC AAC CCA AG - #C TTG GTC CAT GCT CAA          660                                                                       Glu Gly Gly Leu Asp Tyr Leu Gly Asn Pro Se - #r Leu Val His Ala Gln               145              - #   150              - #   155                          - - AGC ATC CTT GCC ATA TGG GCC ACT CAG GTT AT - #C TTG ATG GGA GCT GTC          708                                                                       Ser Ile Leu Ala Ile Trp Ala Thr Gln Val Il - #e Leu Met Gly Ala Val           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - GAA GGT TAC CGT ATT GCC GGT GGG CCT CTC GG - #T GAG GTG GTT GAT        CCA      756                                                                    Glu Gly Tyr Arg Ile Ala Gly Gly Pro Leu Gl - #y Glu Val Val Asp Pro                          180  - #               185  - #               190              - - CTT TAC CCA GGT GGA AGC TTT GAT CCA TTG GG - #C TTA GCT GAT GAT CCA          804                                                                       Leu Tyr Pro Gly Gly Ser Phe Asp Pro Leu Gl - #y Leu Ala Asp Asp Pro                       195      - #           200      - #           205                  - - GAA GCA TTC GCA GAA TTG AAG GTG AAG GAA CT - #C AAG AAC GGT AGA TTA          852                                                                       Glu Ala Phe Ala Glu Leu Lys Val Lys Glu Le - #u Lys Asn Gly Arg Leu                   210          - #       215          - #       220                      - - GCC ATG TTC TCA ATG TTT GGA TTC TTC GTT CA - #A GCT ATT GTA ACT GGA          900                                                                       Ala Met Phe Ser Met Phe Gly Phe Phe Val Gl - #n Ala Ile Val Thr Gly               225              - #   230              - #   235                          - - AAG GGT CCT TTG GAG AAC CTT GCT GAT CAT CT - #T GCA GAC CCA GTC AAC          948                                                                       Lys Gly Pro Leu Glu Asn Leu Ala Asp His Le - #u Ala Asp Pro Val Asn           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - AAC AAT GCA TGG TCA TAT GCC ACC AAC TTT GT - #T CCC GGA AAA                 - # 990                                                                   Asn Asn Ala Trp Ser Tyr Ala Thr Asn Phe Va - #l Pro Gly Lys                                   260  - #               265                                     - - TAAACACTCT TATATTTATA TGTTTTTGTG ATAGTAATCT TCTTCCCAAT TC -             #AATGTGAA   1050                                                                 - - TTATTATCAT TATCATTATC ATGTGGGTAT GCATAGGTTC ACTAATACAA GA -            #TGATGGAT   1110                                                                 - - GCTTTTTTTT TACCAAATTT TAAATTTTAT GTTTCATGCT TTCCATTGCT AG - #ACAT           1166                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 269 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Met Ala Ala Ser Ser Ser Ser Ser Met Ala Le - #u Ser Ser Pro Thr Leu        1               5 - #                 10 - #                 15              - - Ala Gly Lys Gln Leu Lys Leu Asn Pro Ser Se - #r Gln Glu Leu Gly Ala                   20     - #             25     - #             30                  - - Ala Arg Phe Thr Met Arg Lys Ser Ala Thr Th - #r Lys Lys Val Ala Ser               35         - #         40         - #         45                      - - Ser Gly Ser Pro Trp Tyr Gly Pro Asp Arg Va - #l Lys Tyr Leu Gly Pro           50             - #     55             - #     60                          - - Phe Ser Gly Glu Ser Pro Ser Tyr Leu Thr Gl - #y Glu Phe Pro Gly Asp       65                 - # 70                 - # 75                 - # 80       - - Tyr Gly Trp Asp Thr Ala Gly Leu Ser Ala As - #p Pro Gln Thr Phe Ser                       85 - #                 90 - #                 95              - - Lys Asn Arg Glu Leu Glu Val Ile His Ser Ar - #g Trp Ala Met Leu Gly                  100      - #           105      - #           110                  - - Ala Leu Gly Cys Val Phe Pro Glu Leu Leu Se - #r Arg Asn Gly Val Lys              115          - #       120          - #       125                      - - Phe Gly Glu Ala Val Trp Phe Lys Ala Gly Se - #r Gln Ile Phe Ser Glu          130              - #   135              - #   140                          - - Gly Gly Leu Asp Tyr Leu Gly Asn Pro Ser Le - #u Val His Ala Gln Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Leu Ala Ile Trp Ala Thr Gln Val Ile Le - #u Met Gly Ala Val        Glu                                                                                             165  - #               170  - #               175             - - Gly Tyr Arg Ile Ala Gly Gly Pro Leu Gly Gl - #u Val Val Asp Pro Leu                  180      - #           185      - #           190                  - - Tyr Pro Gly Gly Ser Phe Asp Pro Leu Gly Le - #u Ala Asp Asp Pro Glu              195          - #       200          - #       205                      - - Ala Phe Ala Glu Leu Lys Val Lys Glu Leu Ly - #s Asn Gly Arg Leu Ala          210              - #   215              - #   220                          - - Met Phe Ser Met Phe Gly Phe Phe Val Gln Al - #a Ile Val Thr Gly Lys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Pro Leu Glu Asn Leu Ala Asp His Leu Al - #a Asp Pro Val Asn        Asn                                                                                             245  - #               250  - #               255             - - Asn Ala Trp Ser Tyr Ala Thr Asn Phe Val Pr - #o Gly Lys                              260      - #           265                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 221 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION:30..221                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - ACGTTGCAAT TCATACAGAA GTGAGAAAA ATG GCT TCT ATG ATA - # TCC TCT TCC           53                                                                                         - #              Met Ala S - #er Met Ile Ser Ser Ser                          - #              270   - #              275                  - - GCT GTG ACA ACA GTC AGC CGT GCC TCT AGG GG - #G CAA TCC GCC GCA GTG          101                                                                       Ala Val Thr Thr Val Ser Arg Ala Ser Arg Gl - #y Gln Ser Ala Ala Val                   280          - #       285          - #       290                      - - GCT CCA TTC GGC GGC CTC AAA TCC ATG ACT GG - #A TTC CCA GTG AAG AAG          149                                                                       Ala Pro Phe Gly Gly Leu Lys Ser Met Thr Gl - #y Phe Pro Val Lys Lys               295              - #   300              - #   305                          - - GTC AAC ACT GAC ATT ACT TCC ATT ACA AGC AA - #T GGT GGA AGA GTA AAG          197                                                                       Val Asn Thr Asp Ile Thr Ser Ile Thr Ser As - #n Gly Gly Arg Val Lys           310                 3 - #15                 3 - #20                 3 -      #25                                                                              - - TGC ATG GAT CCT GTA GAG AAG TCT     - #                  - #                   221                                                                    Cys Met Asp Pro Val Glu Lys Ser                                                               330                                                            - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - Met Ala Ser Met Ile Ser Ser Ser Ala Val Th - #r Thr Val Ser Arg Ala        1               5 - #                 10 - #                 15              - - Ser Arg Gly Asn Ser Ala Ala Val Ala Pro Ph - #e Gly Gly Leu Lys Ser                   20     - #             25     - #             30                  - - Met Thr Gly Phe Pro Val Lys Lys Val Gln Th - #r Glu Ile Thr Ser Ile               35         - #         40         - #         45                      - - Thr Ser Glu Gly Gly Arg Val Lys Cys Met As - #p Pro Val Glu Lys Ser           50             - #     55             - #     60                        __________________________________________________________________________

We claim:
 1. A DNA construct comprising:a) a promoter; b) a 5'untranslated region containing a translational enhancer; c) DNA encodinga heterologous plastid-specific transit peptide which enhances proteinimport; d) a gene encoding a nuclear-encoded, plastid membrane protein;and e) a 3' untranslated region containing a functional polyadenylationsignal.
 2. The DNA construct according to claim 1 wherein the construct,incorporated into a plant cell or a cell of a photosynthetic organism,enhances photosynthesis or plastid function of the plant cell or thecell of a photosynthetic organism compared to photosynthesis or plastidfunction in a wild-type cell under the same growth conditions.
 3. TheDNA construct according to claim 1, wherein the promoter region is aconstitutive promoter.
 4. The DNA construct according to claim 3,wherein the constitutive promoter is a 35S cauliflower mosaic virus(CaMV) promoter.
 5. The DNA construct according to claim 1, wherein thetranslational enhancer is a translational enhancer of the 5'untranslated region of the small subunit of ribulose-1,5-bisphosphatecarboxylase.
 6. The DNA construct according to claim 5, wherein thetranslational enhancer has a nucleotide sequence comprising residues 1to 29 of SEQ ID NO:3.
 7. The DNA construct according to claim 1, whereinthe transit peptide is from the small subunit ofribulose-1,5-bisphosphate carboxylase.
 8. The DNA construct according toclaim 7, wherein the transit peptide has a nucleotide sequencecomprising residues 30 to 215 of SEQ ID NO:3.
 9. The DNA constructaccording to claim 1, wherein the gene encodes a pigment or apigment-binding protein.
 10. The DNA construct according to claim 1,wherein the gene encodes a chlorophyll a/b binding protein.
 11. The DNAconstruct according to claim 1, wherein the gene encodes a chlorophylla/b binding protein selected from the group consisting of Lhca1, Lhca2,Lhca3, Lhca4, Lhcb1, Lhcb2, Lhcb3, Lhcb4, Lhcb5 and Lhcb6.
 12. The DNAconstruct according to claim 10, wherein the gene encoding a chlorophylla/b binding protein is a pea cab gene.
 13. The DNA construct accordingto claim 7, wherein the 3' untranslated region comprising a functionalpolyadenylation signal is from a cab gene.
 14. A transgenic plant orphotosynthetic organism containing a DNA construct comprising:a) apromoter; b) a 5' untranslated region containing a translationalenhancer; c) DNA encoding a heterologous plastid-specific transitpeptide which enhances protein import; d) a gene encoding anuclear-encoded, plastid membrane protein; and e) a 3' untranslatedregion containing a functional polyadenylation signal.
 15. Seed of thetransgenic plant according to claim
 14. 16. A plant part derived fromthe transgenic plant according to claim 14, wherein the plant partcontains the DNA construct.
 17. The plant part of claim 16 selected fromthe group consisting of leaves, stems, roots, flowers, tissues,epicotyls, meristems, hypocotyls, cotyledons, pollen, ovaries, cells,and protoplasts.
 18. Progeny of the plant or photosynthetic organismaccording to claim 14 wherein the progeny contains the DNA construct.19. The progeny according to claim 18 wherein the gene encodes a pigmentor a pigment-binding protein.
 20. The transgenic plant according toclaim 14 wherein the plant is a monocot.
 21. The transgenic plantaccording to claim 14 wherein the plant is a dicot.
 22. A transgenicplant or photosynthetic organism according to claim 14 wherein the plantor photosynthetic organism further comprises an additional exogenousnucleic acid sequence.
 23. A transgenic plant or photosynthetic organismcontaining a DNA construct comprising:a) a promoter; b) a 5'untranslated region comprising the 5' untranslated region of the smallsubmit of ribulose-1,5-bisphosphate carboxylase; c) DNA encoding aheterologous transit peptide which has a nucleotide sequence comprisingresidues 30-215 of SEQ ID NO:3; d) a gene encoding a nuclear-encodedpigment-binding membrane protein; and e) a 3' untranslated regioncontaining a functional polyadenylation signal.
 24. Seed of thetransgenic plant according to claim
 23. 25. A plant part derived fromthe transgenic plant according to claim 23, wherein the plant partcontains the DNA construct.
 26. The plant part of claim 25 selected fromthe group consisting of leaves, stems, roots, flowers, tissues,epicotyls, meristems, hypocotyls, cotyledons, pollen, ovaries, cells,and protoplasts.
 27. Progeny of the plant or photosynthetic organismaccording to claim 23 wherein the progeny contains the DNA construct.28. The progeny according to claim 27 wherein the gene encodes achlorophyll a/b binding protein.
 29. The transgenic plant according toclaim 23 wherein the plant is a monocot.
 30. The transgenic plantaccording to claim 23 wherein the plant is a dicot.
 31. A transgenicplant or photosynthetic organism according to claim 23 wherein the plantor photosynthetic organism further comprises an additional exogenousnucleic acid sequence.
 32. A cell or tissue culture containing a DNAconstruct comprising:a) a promoter; b) a 5' untranslated regioncontaining a translational enhancer; c) DNA encoding a heterologousplastid-specific transit peptide which enhances protein import; d) agene encoding a nuclear-encoded, plastid membrane protein; and e) a 3'untranslated region containing a functional polyadenylation signal. 33.A plant regenerated from the cell or tissue culture according to claim32.
 34. A cell or tissue culture according to claim 32 containing anadditional exogenous nucleic acid sequence.
 35. A plant regenerated fromthe cell or tissue culture according to claim
 34. 36. A method forenhancing the photosynthetic or biosynthetic capability of a plant,tissue culture or photosynthetic organism comprising:a) preparing a DNAconstruct comprising a promoter, a 5' untranslated region containing atranslational enhancer, DNA encoding a heterologous plastid-specifictransit peptide which enhances protein import, a gene encoding anuclear-encoded plastid membrane protein, and a 3' untranslated regioncontaining a functional polyadenylation signal; b) inserting the DNAconstruct into a cloning vector; and c) transforming a plant, tissueculture or photosynthetic organism with the cloning vector.
 37. A methodfor detecting transformation in plants, plant tissue or a photosyntheticorganism consisting of:a) preparing a DNA construct comprising apromoter, a 5' untranslated region containing a translational enhancer,DNA encoding a heterologous plastid-specific transit peptide whichenhances protein import, a gene encoding a nuclear-encoded plastidmembrane protein the expression of which is detectable, and a 3'untranslated region containing a functional polyadenylation signal; b)inserting the DNA construct into a cloning vector; and c) transforming aplant, tissue culture or photosynthetic organism with the cloning vectorso that the protein is expressed,wherein expression of the protein isindicative of transformation.
 38. The method of claim 37 wherein theprotein is a pigment or pigment-binding molecule.
 39. The method ofclaim 38 wherein the protein is a chlorophyll a/b binding protein. 40.The method according to claim 39 wherein expression, in comparison towild-type plants under the same conditions, is manifested by one or moreof the following characteristics: increased yield, enhancedpigmentation, increased biomass, increased carbohydrate content, moreuniform growth, larger seeds or fruits, increased stem girth, enhancedphotosynthesis under low light conditions, faster germination, andincreased ability to withstand transplant shock.